THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board GARY N. CALKINS, Columbia University E. E. JUST, Howard University E. G. CONKLIN, Princeton University FRANK R. LlLLIE, University of Chicago 1LS: 5tV P f nCe K t0n TT UniVerSity CARL R ' MOORE, University of Chicago SELIG HECHT, Columbia University ~ _ T.- LEIGH HOADLEY, Harvard University GEORGE T. MOORE, Missouri Botanical Garden L. IRVING, Swarthmore College T. H. MORGAN, California Institute of Technology M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University H. S. JENNINGS, Johns Hopkins University F. SCHRADER, Columbia University ALFRED C. REDFIELD, Harvard University Managing Editor VOLUME LXXVII AUGUST TO DECEMBER, 1939 Printed and Issued by LANCASTER PRESS, Inc. PRINCE &. LEMON STS. LANCASTER, PA. 11 THE BIOLOGICAL BULLETIN is issued six times a year. Single numbers, $1.75. Subscription per volume (3 numbers), $4.50. Subscriptions and other matter should be addressed to the Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa. Agent for Great Britain : Wheldon & Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W.C. 2. Communications relative to manuscripts should be sent to the Managing Editor, Marine Biological Laboratory, Woods Hole, Mass., between June 1 and October 1 and to the Biological Labo- ratories, Divinity Avenue, Cambridge, Mass., during the remainder of the year. Entered October 10, 1902, at Lancaster, Pa., as second-class matter under Act of Congress of July 16, 1894. LANCASTER PRESS, INC., LANCASTER, PA. CONTENTS No. 1. A.UGUST, 1939 PAGE FORTY-FIRS^ ^ x ^INE BIOLOGICAL LABORATORY. 1 irematodes of Woods Hole. II. The life o "' .. " - 01 Stephanostomum tenue (Linton) 65 IIARVEY, ETHEL BROWNE An Hermaphrodite Arbacia 74 ROOSEN-RUNGE, EDWARD C. Karyokinesis during Cleavage of the Zebra fish Brachydanio rerio 79 MATTHEWS, SAMUEL A. The Effects of Light and Temperature on the Male Sexual Cycle in Fundulus 92 BURGER, J. WENDELL Some Experiments on the Relation of the External Environ- ment to the Spermatogenetic Cycle of Fundulus heteroclitus (L.) 96 BROWN, F. A., JR., AND ONA CUNNINGHAM Influence of the Sinusgland of Crustaceans on Normal Via- bility and Ecdysis 104 MACGINITIE, G. E. The Method of Feeding of Chaetopterus 115 WELSH, JOHN H. The Action of Eye-stalk Extracts on Retinal Pigment Migra- tion in the Crayfish, Cambarus bartoni 119 CROZIER, W. J., AND ERNST WOLF The Flicker-response Contour for the Crayfish. II. Retinal pigment and the theory of the asymmetry of the curve 126 LAWSON, CHESTER A. The Significance of Germaria in Differentiation of Ovarioles in Female Aphids 135 51145 111 11 THE BIOLOGICAL BULLETIN is issued six times a year. Single numbers, $1.75. Subscription per volume (3 numbers), $4.50. Subscriptions and other matter shou) v addressed to the Biological Bulletin, Prince and Lemon Lancaster, Pa. Agent for Great Britain: Wheldon & \v A . 2, 3 and 4 Arthur Street, New Oxford Street, London, Communications relative to manuscripts should ,^ Managing Editor, Marine Biological Laboratory, Woo. Mass., between June 1 and October 1 and to the Biological i^. ratories, Divinity Avenue, Cambridge, Mass., during the remaindei of the year. Entered October 10, 1902, at Lancaster, Pa., as second-class matter under Act of Congress of July 16, 1894. LANCASTER PRESS, INC., LANCASTER, PA. CONTENTS No. 1. AUGUST, 1939 PAGE FORTY- FIRST REPORT OF THE MARINE BIOLOGICAL LABORATORY. 1 MARTIN, W. E. Studies on the Trematodes of Woods Hole. II. The life cycle of Stephanostomum tenue (Linton) 65 HARVEY, ETHEL BROWNE An Hermaphrodite Arbacia 74 ROOSEN-RUNGE, EDWARD C. Karyokinesis during Cleavage of the Zebra fish Brachydanio rerio 79 MATTHEWS, SAMUEL A. The Effects of Light and Temperature on the Male Sexual Cycle in Fundulus 92 BURGER, J. WENDELL Some Experiments on the Relation of the External Environ- ment to the Spermatogenetic Cycle of Fundulus heteroclitus (L.) 96 BROWN, F. A., JR., AND ONA CUNNINGHAM Influence of the Sinusgland of Crustaceans on Normal Via- bility and Ecdysis 104 MACGINITIE, G. E. The Method of Feeding of Chaetopterus 115 WELSH, JOHN H. The Action of Eye-stalk Extracts on Retinal Pigment Migra- tion in the Crayfish, Cambarus bartoni 119 CROZIER, W. J., AND ERNST WOLF The Flicker-response Contour for the Crayfish. II. Retinal pigment and the theory of the asymmetry of the curve 126 LAWSON, CHESTER A. The Significance of Germaria in Differentiation of Ovarioles in Female Aphids 135 51145 111 iv CONTENTS No. 2. OCTOBER, 1939 PAGE SOUTHWICK, WALTER E. Activity-preventing and Egg-Sea-Water Neutralizing Sub- stances from Spermatozoa of Echinometra subangularis .... 147 SOUTHWICK, WALTER E. The "Agglutination" Phenomenon with Spermatozoa of Chiton tuberculatus 157 KANDA, SAKYO The Luminescence of a Nemertean, Emplectonema kandai, Kato 166 FAWCETT, DON WAYNE Absence of the Epithelial Hypophysis in a Fetal Dogfish Associated with Abnormalities of the Head and of Pigmenta- tion 174 GOODRICH, H. B., AND PRISCILLA L. ANDERSON Variations of Color Pattern in Hybrids of the Goldfish, Carassius auratus 184 GOODRICH, H. B., AND J. P. TRINKAUS The Differential Effect of Radiations on Mendelian Pheno- types of the Goldfish, Carassius auratus 192 JOHNSON, W. H., AND J. E. G. RAYMONT The Reactions of the Planktonic Copepod, Centropages typicus, to Light and Gravity 200 ROSE, S. MERYL Embryonic Induction in the Ascidia 216 PORTER, K. R. Androgenetic Development of the Egg of Rana pipiens 233 BUTCHER, EARL O. The Illumination of the Eye Necessary for Different Melano- phoric Responses of Fundulus heteroclitus 258 BRAGG, ARTHUR N. Observations upon Amphibian Deutoplasm and its Relation to Embryonic and Early Larval Development 268 VON BRAND, THEODOR, NORRIS W. RAKESTRAW AND CHARLES E. RENN Further Experiments on the Decomposition and Regeneration of Nitrogenous Organic Matter in Sea Water 285 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1939. 297 CONTENTS v No. 3. DECEMBER, 1939 PAGE KlTCHING, J. A. The Effects of a Lack of Oxygen and of Low Oxygen Tensions on Paramecium 339 RAYMONT, J. E. G. Dark Adaptation and Reversal of Phototropic Sign in Dineutes 354 BlSSONNETTE, THOMAS HUME AND ALBERT GEORGE CSECH Modified Sexual Photoperiodicity in Cotton-tail Rabbits . . . 364 LlTTLEFORD, ROBERT A. The Life Cycle of Dactylometra quinquecirrha, L. Agassiz in the Chesapeake Bay 368 BROWN, MORDEN G. The Blocking of Excystment Reactions of Colpoda duo- denaria by Absence of Oxygen 382 MAST, S. O. The Relation between Kind of Food, Growth, and Structure in Amoeba 391 ANGERER, C. A. The Effect of Electric Current on the Relative Viscosity of Sea-Urchin Egg Protoplasm 399 BEADLE, G. W., E. L. TATUM AND C. W. CLANCY Development of Eye Colors in Drosophila: Production of v + Hormone by Fat Bodies 407 TATUM, E. L., AND G. W. BEADLE Effect of Diet on Eye-Color Development in Drosophila melanogaster 415 RUSSELL, ALICE Pigment Inheritance in the Fundulus-Scomber Hybrid 423 CHILD, GEORGE The Effect of Increasing Time of Development at Constant Temperature on the Wing Size of Vestigial of Drosophila melanogaster 432 MACGINITIE, G. E. The Method of Feeding of Tunicates 443 DEWEY, VIRGINIA C. Test Secretion in Two Species of Folliculina 448 INDEX FOR VOLUME 77 . 457 Vol. LXXVII, No. 1 August, 1939 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE MARINE BIOLOGICAL LABORATORY FORTY-FIRST REPORT, FOR THE YEAR 1938 FIFTY-FIRST YEAR I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 9, 1938) 1 STANDING COMMITTEES 3 II. ACT OF INCORPORATION 3 III. BY-LAWS OF THE CORPORATION 4 IV. REPORT OF THE TREASURER 5 V. REPORT OF THE LIBRARIAN 10 VI. REPORT OF THE DIRECTOR 11 Statement 11 Addenda : 1. Report of the Committee on Policy 15 2. The Staff, 1938 27 3. Investigators and Students, 1938 30 4. Tabular View of Attendance 41 5. Subscribing and Cooperating Institutions, 1938 .... 41 6. Evening Lectures, 1938 42 7. Shorter Scientific Papers, 1938 43 8. General Scientific Meeting, 1938 45 9. Members of the Corporation 50 I. TRUSTEES EX OFFICIO FRANK R. LILLIE, President of the Corporation, The University of Chicago. CHARLES PACKARD, Associate Director, Columbia University. LAWRASON RIGGS, JR., Treasurer, 120 Broadway, New York City. PHILIP H. ARMSTRONG, Clerk of the Corporation, Syracuse University and Medical College. EMERITUS H. C. BUMPUS, Brown University. E. G. CONKLIN, Princeton University. C. R. CRANE, New York City. R. A. HARPER, Columbia University. H. S. JENNINGS, Johns Hopkins University. M. M. METCALF, Waban, Mass. T. H. MORGAN, California Institute of Technology. I MARINE BIOLOGICAL LABORATORY G. H. PARKER, Harvard University. W. B. SCOTT, Princeton University. E. B. WILSON, Columbia University. TO SERVE UNTIL 1942 E. R. CLARK, University of Pennsylvania. OTTO C. GLASER, Amherst College. Ross G. HARRISON, Yale University. E. N. HARVEY, Princeton University. M. H. JACOBS, University of Pennsylvania. F. P. KNOWLTON, Syracuse University. FRANZ SCHRADER, Columbia University. B. H. WILLIER, University of Rochester. TO SERVE UNTIL 1941 W. R. AMBERSON, University of Tennessee. W. C. CURTIS, University of Missouri. H. B. GOODRICH, Wesleyan University. I. F. LEWIS, University of Virginia. R. S. LILLIE, The University of Chicago. A. C. REDFIELD, Harvard University. C. C. SPEIDEL, University of Virginia. D. H. TENNENT, Bryn Mawr College. TO SERVE UNTIL 1940 H. B. BIGELOW, Harvard University. R. CHAMBERS, Washington Square College, New York University. W. E. GARREY, Vanderbilt University Medical School. CASWELL GRAVE, Washington University. S. O. MAST, Johns Hopkins University. A. P. MATHEWS, University of Cincinnati. C. E. McCLUNG, University of Pennsylvania. C. R. STOCKARD, Cornell University Medical College. TO SERVE UNTIL 1939 W. C. ALLEE, The University of Chicago. GARY N. CALKINS, Columbia University. B. M. DUGGAR, University of Wisconsin. L. V. HEILBRUNN, University of Pennsylvania. L. IRVING, University of Toronto. W. J. V. OSTERHOUT, Member of the Rockefeller Institute for Medical Re- search. A. H. STURTEVANT, California Institute of Technology. LORANDE L. WOODRUFF, Yale University. EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES FRANK R. LILLIE, Ex. Off. Chairman. CHARLES PACKARD, Ex. Off. LAWRASON RIGGS, JR., Ex. Off. CASWELL GRAVE, to serve until 1939. C. E. MCCLUNG, to serve until 1939. ACT OF INCORPORATION LAURENCE IRVING, to serve until 1940. S. O. MAST, to serve until 1940. THE LIBRARY COMMITTEE E. G. CON KLIN, Chairman. WILLIAM R. AMBERSON. C. O. ISELIN, II. C. C. SPEIDEL. A. H. STURTEVANT. WILLIAM R. TAYLOR. THE APPARATUS COMMITTEE L. V. HEILBRUNN, Chairman. W. R. AMBERSON. D. J. EDWARDS. W. E. CARREY. E. N. HARVEY. L. IRVING. M. H. JACOBS. B. LUCKE. THE SUPPLY DEPARTMENT COMMITTEE LAURENCE IRVING, Chairman. T. H. BlSSONNETTE. H. B. GOODRICH. A. C. REDFIELD. C. C. SPEIDEL. THE EVENING LECTURE COMMITTEE B. H. WILLIER, Chairman. M. H. JACOBS. CHARLES PACKARD. II. ACT OF INCORPORATION No. 3170 COMMONWEALTH OF MASSACHUSETTS Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedg- wick Minot, Samuel Wells, William G. Farlow, Anna D. Phillips and B. H. Van Vleck have associated themselves with the intention of forming a Corporation under the name of the Marine Biological Laboratory, for the purpose of establishing and maintaining a laboratory or station for scien- tific study and investigation, and a school for instruction in biology and natural history, and have complied with the provisions of the statutes of this Commonwealth in such case made and provided, as appears from the cer- tificate of the President, Treasurer, and Trustees of said Corporation, duly approved by the Commissioner of Corporations, and recorded in this office; Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachusetts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardiner, "S. Minns, C. S. Minot, S. Wells, W. 4 MARINE BIOLOGICAL LABORATORY G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and suc- cessors, are legally organized and established as, and are hereby made, an existing Corporation, under the name of the MARINE BIOLOGICAL LABORATORY, with the powers, rights, and privileges, and subject to the limitations, duties, and restrictions, which by law appertain thereto. Witness my official signature hereunto subscribed, and the seal of the Commonwealth of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord One Thousand Eight Hundred and Eighty- Eight. [SEAL] HENRY B. PIERCE, Secretary of the Commonwealth. III. BY-LAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY I. The annual meeting of the members shall be held on the second Tuesday in August, at the Laboratory, in Woods Hole, Mass., at 11.30 A.M., daylight saving time, in each year, and at such meeting the members shall choose by ballot a Treasurer and a Clerk to serve one year, and eight Trustees to serve four years. There shall be thirty-two Trustees thus chosen divided into four classes, each to serve four years, and in addition there shall be two groups of Trustees as follows: (a) Trustees ex officio, who shall be the President of the Corporation, the Director of the Laboratory, the Associate Director, the Treasurer and the Clerk; (&) Trustees Emeritus, who shall be elected from the Trustees by the Corporation. Any regular Trustee who has attained the age of seventy years shall continue to serve as Trustee until the next annual meeting of the Corporation, whereupon his office as regular Trustee shall become vacant and be filled by election by the Cor- poration and he shall become eligible for election as Trustee Emeritus for life. The Trustees ex officio and Emeritus shall have all rights of the Trustees except that Trustees Emeritus shall not have the right to vote. The Trustees and officers shall hold their respective offices until their successors are chosen and have qualified in their stead. II. Special meetings of the members may be called by the Trustees to be held in Boston or in Woods Hole at such time and place as may be designated. III. Inasmuch as the time and place of the Annual Meeting of Members is fixed by these By-laws, no notice of the Annual Meeting need be given. Notice of any special meeting of members, however, shall be given by the Clerk by mailing notice of the time and place and purpose of said meeting, at least fifteen (15) days before such meeting, to each member at his or her address as shown on the records of the Corporation. IV. Twenty-five members shall constitute a quorum at any meeting. V. The Trustees shall have the control and management of the affairs of the Corporation; they shall present a report of its condition at every annual meeting; they shall elect one of their number President of the Cor- poration who shall also be Chairman of the Board of Trustees; they shall appoint a Director of the Laboratory; and they may choose such other officers and agents as they may think best; they may fix the compensation and REPORT OF THE TREASURER define the duties of all the officers and agents; and may remove them, or any of them, except those chosen by the members, at any time; they may fill vacancies occurring in any manner in their own number or in any of the offices. They shall from time to time elect members to the Corporation upon such terms and conditions as they may think best. VI. Meetings of the Trustees shall be called by the President, or by any two Trustees, and the Secretary shall give notice thereof by written or printed notice sent to each Trustee by mail, postpaid. Seven Trustees shall constitute a quorum for the transaction of business. The Board of Trustees shall have power to choose an Executive Committee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient. VII. The accounts of the Treasurer shall be audited annually by a certified public accountant. VIII. The consent of every Trustee shall be necessary to dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such manner and upon such terms as shall be de- termined by the affirmative vote of two-thirds of the Board of Trustees. IX. These By-laws may be altered at any meeting of the Trustees, pro- vided that the notice of such meeting shall state that an alteration of the By-laws will be acted upon. X. Any member in good standing may vote at any meeting, either in person or by proxy duly executed. IV. THE REPORT OF THE TREASURER To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY: Gentlemen: Herewith is my report as Treasurer of the Marine Biological Laboratory for the year 1938. The accounts have been audited by Messrs. Seamans, Stetson and Tuttle, certified public accountants. A copy of their report is on file at the Laboratory and is open to inspection by members of the Cor- poration. At the end of the year 1938, the book value of the Endowment Funds in the hands of the Central Hanover Bank and Trust Company as Trustee, was General Fund, Securities (market $862,409.23) $ 916,855.70 9,235.71 858.45 173,918.24 20,102.88 319.46 Real Estate Cash, principal Library Fund, Securities (market $162,008.68) Real Estate Cash $1,121,290.44 - ash. 6 MARINE BIOLOGICAL LABORATORY The income collected from these Funds was as follows : General Endowment $36,382.94 Library 6,665.66 $43,048.60 The income in arrears on these Funds at the end of the year was : Arrears General Fund $13,518.86 Arrears Library Fund 3,450.00 $16,968.86 Arrears at the end of the year 1937 $12,755.86 showing an increase of $ 4,213.00 The dividends from the General Biological Supply House totalled $14,224.00. Retirement Fund: A total of $4,060 was paid in pensions of which $197.20 was advanced from current funds. The Fund at the end of the year consisted of mortgages and real estate at the book value of $17,462.08. Plant Assets: The land (exclusive of Gansett and Devil's Lane), buildings, equipment and library represent an investment of $1,789,884.74 less reserve for depreciation 517,178.00 or a net of $1,272,706.74 The hurricane water damage to the inventory and equipment and plant amounted to $30,399.02 of which $2,387.97 was charged to Plant Fund and $28,011.05 to Cur- rent Surplus. Early this year The Carnegie Corporation of New York most generously contributed $20,000 toward the repair of the hurricane damage. Income and Expenses: Income including a donation of stock valued at $7,250 exceeded expense, including $24,481.56 depreciation, by $11,432.64. There was expended from current funds for plant account a net of $15,083.21 and in addition $6,500 in reduction of mortgage and note indebtedness. At the end of the year the Laboratory owed $5,500 on mortgages and $7,000 on notes all for property purchased in earlier years. It had accounts and. notes receivable of $12,305.69 and $8,531.29 in cash and bank accounts in its current funds. REPORT OF THE TREASURER 7 A gift of 200 shares of Crane Company stock was received from Dr. Frank R. Lillie, to which he has since added 300 shares. Following is the balance sheet, the condensed statement of income and outgo, and the surplus account all as set out by the accountants : EXHIBIT A MARINE BIOLOGICAL LABORATORY BALANCE SHEET, DECEMBER 31, 1938 Assets Endowment Assets and Equities : Securities and Cash in Hands of Central Hanover Bank and Trust Company, New York, Trustee Schedules I-a and I-b $1,121,290.44 Securities and Cash Minor Funds Schedule II .. 8,742.81 $1,130,033.25 Plant Assets : Land Schedule IV $ 110,884.58 Buildings Schedule IV 1,239,161.81 Equipment Schedule IV 165,567.34 Library Schedule IV 274,271.01 $1,789,884.74 Less Reserve for Depreciation 517,178.00 $1,272,706.74 Cash in Dormitory Building Fund 223.24 Cash in Reserve Fund 24.65 $1,272,954.63 Current Assets : Cash $ 8,531.29 Accounts and Notes Receivable 12,305.69 Inventories : Supply Department $ 37,672.27 Biological Bulletin 9,762.64 47,434.91 Investments : Devil's Lane Property $ 44,398.34 Gansett Property 5,822.49 Stock in General Biological Supply House, Inc 12,700.00 Other Investment Stocks 7,250.00 Securities and Real Estate Re- tirement Fund List Sched- ule V, viz., Retirement Fund Por- tion 17,264.88 Current Account Portion . 197.20 87,632.91 Prepaid Insurance 3,193.90 Items in Suspense (Net) 693.98 $ 159,792.68 $2,562,780.56 8 MARINE BIOLOGICAL LABORATORY Liabilities Endowment Funds : Endowment Funds Schedule III $1,120,581.61 Reserve for Amortization of Bond Premiums 708.83 $1,121,290.44 Minor Funds Schedule III 8,742.81 $1,130,033.25 Plant Liabilities and Funds : Mortgage Payable, Howes Property $ 5,500.00 Notes Payable a/c Bar Neck Property Purchase . 7,000.00 Donations and Gifts Schedule III 1,038,402.61 Other Investments in Plant from Gifts and Cur- rent Funds 222,052.02 $1,272,954.63 Current Liabilities and Surplus : Accounts Payable Reserve for Additional Repairs and Replacements on account of Hurricane Water Damage Current Surplus Exhibit C EXHIBIT B 4,077.37 17,518.12 138,197.19 159,792.68 $2,562,780.56 MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE, YEAR ENDED DECEMBER 31, 1938 Total Net Expense Income Expense Income Income : General Endowment Fund $ 36,382.94 $ 36,382.94 Library Fund 6,665.66 6,665.66 Donations 7,250.00 7,250.00 Instruction 8,356.14 9,960.00 1,603.86 Research 4,215.07 16,312.50 12,097.43 Evening Lectures 58.56 58.56 Biological Bulletin and Membership Dues 9,691.33 10,362.75 671.42 Supply Department Schedule VI . 40,814.01 38,134.93 2,679.08 Mess Schedule VII 25,899.67 25,759.83 139.84 Dormitories Schedule VIII 22,609.70 12,973.34 9,636.36 ( Interest and Depreciation charged to above 3 Departments See Schedules VI, VII, and VIII) 23,731.15 23,731.15 Dividends, General Biological Sup- ply House, Inc 14,224.00 14,224.00 Rents : Bar Neck Property 3,568.46 3,568.46 Bay Shore Property 206.57 91.75 114.82 Howes Property 196.64 480.00 283.36 Janitor House 23.19 360.00 336.81 Newman Cottage 81.43 250.00 168.57 Danchakoff Cottage 324.30 750.00 425.70 REPORT OF THE TREASURER Sale of Library Duplicates 390.73 390.73 Apparatus Rental 991.30 991.30 Interest on Notes Receivable 150.00 150.00 Sundry Income 38.54 38.54 Maintenance of Plant : Buildings and Grounds 22,482.48 22,482.48 Chemical and Special Apparatus Expense 14,121.00 14,121.00 Library Expense 7,576.77 7,576.77 Truck Expense 1,249.70 1,249.70 Workmen's Compensation Insurance 507.51 507.51 Sundry Expense 19.50 19.50 General Expenses : Administration Expense 12,199.94 12,199.94 Endowment Fund Trustee and Safe-keeping 1,001.95 1,001.95 Interest on Notes and Mortgage -Payable 829.83 829.83 Bad Debts 448.39 448.39 Reserve for Depreciation 24,481.56 24,481.56 $173,664.09 $185,096.73 $ 97,547.29 $108,979.93 Excess of income over Expense carried to Current Surplus- Exhibit C 11,432.64 11,432.64 $185,096.73 $108,979.93 EXHIBIT C MARINE BIOLOGICAL LABORATORY, CURRENT SURPLUS ACCOUNT, YEAR ENDED DECEMBER 31, 1938 Balance, January 1, 1938 $153,266.82 Add: Excess of Income over Expense for Year as shown in Exhibit B $11,432.64 Reserve for Depreciation Charged to Plant Funds 24,481.56 35,914.20 $189,181.02 Deduct : Payments from Current Funds during Year for Plant Assets as shown in Schedule IV, Buildings $ 939.80 Equipment 4,818.61 Library 9,499.80 $15,258.21 Less Received for Plant Assets Disposed of 175.00 $15,083.21 Payment on Plant Mortgage and Note Payable $ 4,500.00 Pensions Paid $4,060.00 10 MARINE BIOLOGICAL LABORATORY Expenses on Account of Retirement Fund Securities 36.79 $4,096.79 Less Retirement Fund Income and Gain from Security Sale 707.22 3,389.57 Hurricane Water Damage (except portion Charged to Plant Funds) 28,011.05 50,983.83 Balance, December 31, 1938 Exhibit A $138,197.19 Respectfully submitted, LAWRASON RIGGS, JR., Treasurer. V. THE REPORT OF THE LIBRARIAN A report of the expenditures from the $18,800, appropriated to the Library in 1938, follows: books, $351.67; current serials, $5,319.86; binding, $1,171.38 ($45.00 of this on insurance); express, $181.48; supplies, $1,070.07 (includes $37.53 for new boxes to ship books to the bindery; $707.73 for new catalogue cases); salaries, $7,150.00; back sets, $1,795.33; total, $17,039.79. For various reasons such as lack of space in the Library and the difficulty of securing the present lacks except in Germany, where prices are high, it seemed best to allow the $1,760.21 available for back sets besides $390.73 for the Library sale of duplicates, to revert to the General Fund of the Laboratory. Also a correction of the printed 1937 report must be made here. An order for the back set of " Flora " placed in Germany failed to come through and the order was finally cancelled by the Librarian, allowing another sum of $2,430.50 to drop from the Library expenditures. The usual appropriation to the Library of $600.00 by the Woods Hole Oceanographic Institution was expended to the amount of $591.98 and separately accounted. This year the Library lists but 1,306 current serials of which 426 are subscriptions, 385 (11 new) purchases of the Marine Biological Laboratory, 41 (1 new) of the Woods Hole Oceanographic Institution; 666 are exchanges, 596 (4 new) with the BIOLOGICAL BULLETIN and 70 (1 new) with the Woods Hole Oceanographic Institution publica- tions; and 207 come as gifts to the former and 7 as gifts to the latter. The record shows 47 books purchased, 41 by the Marine Biological Laboratory and 6 by the Woods Hole Oceanographic Institution, 19 presented by the authors and 41 from publishers; while a contribution from Dr. Alfred Meyer enabled the Library to purchase a new " Ameri- REPORT OF THE DIRECTOR can Medical Directory"; and Dr. Douglas M. Whitaker presented a copy of Beaumont's " Experiments and Observations on the Gastric Juice and the Physiology of Digestion." Completed back sets of serials number 36; as purchases of the Marine Biological Laboratory, 20, of the Woods Hole Oceanographic Institution, 2 ; while purchases partially completing back sets number 15 for the former and 1 for the latter; through exchange of duplicates, 11 completed back sets for the former, and 1 for the latter ; besides many additions to still incomplete back sets ; and 2 sets for the Marine Biological Laboratory completed by gifts, with 4 partially completed. Reprint additions number 6,905 : current for 1937, 1,897; current for 1938, 894, and of date previous to 1937, 4,114; about 200 of the latter kindly presented by Dr. M. A. Bigelow and 70 by Mrs. H. H. Donaldson. A summary of the current holdings of the Library proper is therefore 44,897 bound volumes and 108,927 reprints. VI. THE REPORT OF THE DIRECTOR To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY : Gentlemen: I beg to present herewith a report of the fifty-first session of the Marine Biological Laboratory for the year 1938. 1. Attendance. The number of investigators and their assistants present during the summer of 1938 was somewhat less than in 1937, but it taxed the facilities of the Laboratory to the utmost. Attendance has risen steadily, with minor fluctuations, since 1933 ; now the number present is greater than the optimum which can be cared for under existing conditions. We are rapidly approaching the time when selec- tion among the applicants for research space must be made, a situation referred to in the report of the Committee on Future Policy in the following words : " It will be necessary to adopt more definite policies concerning the admission of investigators than in the past. These should not, however, be of too binding a character, but rather a definition of principles within which the Director will have free scope for the exercise of his best judgment." The definition of these principles deserves the most careful consideration. 2. The Library. The continued growth of the Library is a source of satisfaction to the investigator, but it presents a serious problem to the Librarian who must find a place for new volumes and reprints. Each year's increment of bound volumes requires a space about equal to one complete stack. Since the present stacks are already practically filled, it will presently be necessary to use rooms now employed for cataloguing or other Library purposes. This will disturb the present orderly arrangement of serials and will at best provide only temporary relief. An addition to the Library is urgently needed. c (LI v 12 MARINE BIOLOGICAL LABORATORY 3. The Board of Trustees. At the meeting of the Corporation held Tuesday, August 9, 1938, Dr. H. S. Jennings, Trustee since 1905, was elected Trustee Emeritus. To fill his place in the Class of 1942, Dr. M. H. Jacobs, the retiring Director, was chosen. At the same meeting, Dr. P. H. Armstrong was elected Clerk of the Corporation in place of Dr. Charles Packard who resigned when appointed Assistant Director. The Board has suffered heavy losses by death. Mr. Charles R. Crane, Trustee from 1901 and President of the Board from 1902 to 1925, " the best friend the Laboratory ever had " ; Dr. Edmund B. Wilson, Trustee continuously from 1890, whose contributions from this Laboratory were instrumental in establishing its scientific eminence ; Dr. Charles R. Stockard, Trustee from 1920, whose counsels, vigor- ously expressed, were always highly valued ; and Dr. J. Playf air Mc- Murrich, Trustee from 1892 to 1900, active in the early days of this Laboratory. 4. The Hurricane and Flood. We may be profoundly thankful that in the storm of September 21, 1938 no one connected with the Laboratory lost his life. Some were rescued from desperate situations, and many suffered heavy material loss. Damage to Laboratory property was due almost entirely to water which poured into the basement of the Brick Building, into the Supply Department and the Dormitory, washed away most of the foundations of the Club House, and carried the Bathhouse far inland. The old laboratory buildings and the Mess were above the flood level. During the height of the storm our staff worked heroically to protect the buildings and equipment. Mr. Larkin organized a bucket brigade and saved the apparatus in the Pump House ; Mr. Mclnnis and his crew protected the motor boats ; others barricaded doors in the Brick Build- ing against the rising waters, but to no avail for the flood broke through the windows in the sub-basement of the Library; Mr. MacNaught opened the Apartment House to those who had been driven from their homes. The greatest loss occurred in the Brick Building where the water, four feet deep, submerged microscopes and electrical apparatus, overran the storage battery, the switchboard and motors, and reduced the chemi- cal and storage rooms to utter confusion. The work of repair began at once. Dr. Pond and his assistants examined all the apparatus which had been wet with salt water, recon- ditioned much of it in our workship, and sent some to the manufacturers for servicing. To restore the Chemical Room required many weeks of hard work. Mr. Mclnnis and his men quickly reduced the confusion in the Supply Department where the damage was not great, and were REPORT OF THE DIRECTOR 13 able, within a few days, to resume regular business. Under the direc- tion of Mr. Larkin, the storage battery was cleaned and recharged, and the various motors were dried and set in place. None were lost, but some needed repairs. The switchboard was damaged but has been re- conditioned. Mrs. Montgomery saved many of the more important duplicate reprints which had been water-soaked. Fortunately the regu- lar reprint collection and the bound volumes were never in danger. The bathhouse, after being put back on new foundations, was damaged by a second storm. By order of the Executive Committee it was removed entirely. These very extensive repairs to the buildings and the equipment have been made almost entirely by our permanent staff who have given un- sparingly of their time and energy. To them the Laboratory owes a debt of gratitude. In the Treasurer's Report the loss due to the storm is set at $30,400. This sum includes all of the various items which were lost. Inasmuch as many of these were of little actual value, and need not be replaced, the actual cost of restoring the damage will undoubtedly be less than $25,000. Since the Laboratory carried no insurance against this type of loss, the financial burden thus imposed upon us was serious. But we are fortunate in our friends. The Carnegie Corporation of New York, a benefactor of former years, has presented to the Laboratory the sum of $20,000 to be used for purposes of restoration. We are sincerely grateful for this generous and timely gift. 5. Research in Botany. For some years it has been apparent that the number of investigators at the Laboratory carrying on research in Botany has declined. This situation is due in part to the fact that some of the members of the Research Staff have been unable to attend the summer session, and in part to the lack of facilities for pursuing re- search in the dynamic phases of Botany. Following the resignation of Drs. Ivy M. Lewis, C. E. Allen and W. J. Robbins from the staff after many years of active service, Dr. E. W. Sinnott, of Columbia University, and Dr. D. R. Goddard, of the University of Rochester, were appointed. The lack of facilities for research has been stressed by many botanists who have expressed the opinion that more laboratory space is needed, that a suitable plot of ground for raising plants should be provided, and that a greenhouse is an essential part of an active botanical laboratory. These requirements should be met at the earliest opportunity. 6. Gifts. The .sum of $20,000 given by the Carnegie Corporation of New York, to be used for the purpose of restoring the damage done by the flood, has already been mentioned. The Marine Biological Labora- tory also gratefully acknowledges gifts amounting to $17,775 presented by Dr. F. R. Lillie. 14 MARINE BIOLOGICAL LABORATORY 7. The Committee on Future Policy. At the meeting of August 11, 1937, the Board of Trustees authorized the President to appoint a com- mittee to formulate a statement concerning the policies and future of the Marine Biological Laboratory. The members of this Committee are : E. G. Conklin, Chairman, G. N. Calkins, W. C. Curtis, H. B. Goodrich, M. H. Jacobs, T. H. Morgan, G. H. Parker, A. C. Redfield and C. R. Stockard. After many discussions during the summers of 1937 and 1938 a report was drawn up by Dr. Lillie. This was studied and amended by the Committee and is now presented on p. 15 of this Annual Report. 8. Lectures and Scientific Meetings. During the summer of 1938 there were ten regular evening lectures and seven seminars at which shorter papers were discussed. In addition to these there were several informal exhibitions of motion pictures of scientific interest and a number of discussion groups. At the final scientific meetings, held August 30 and August 31, numerous investigators reported the results of their work during the current summer. In addition, many demon- strations were on display, both at the Laboratory and at the Fish Com- mission. One of the regular seminar evenings was devoted to an informal celebration of the fiftieth anniversary of the founding of the Laboratory. Dr. Conklin reviewed the history of the early days, and Dr. Lillie spoke of those who have contributed to the scientific and material welfare of the institution. At the close of the meeting he presented to the Labora- tory, in behalf of the Trustees, a portrait of Mr. Crane. It was a great source of satisfaction that Mr. Crane could be present to receive greet- ings from his many friends. As in previous years, the Laboratory was host to the Genetics So- ciety of America, which held its meetings on August 31 and Sep- tember 1. There are appended as parts of the report : 1. The Report of the Committee on Policies and Future of the Marine Biological Laboratory. 2. The Staff, 1938. 3. Investigators and Students, 1938. 4. A Tabular View of Attendance, 1934-38. 5. Subscribing and Cooperating Institutions, 1938. 6. Evening Lectures, 1938. 7. Shorter Scientific Papers, 1938. 8. General Scientific Meeting, 1938. 9. Members of the Corporation, 1938. Respectfully submitted, CHARLES PACKARD, Associate Director. REPORT OF THE DIRECTOR 15 1. REPORT OF THE COMMITTEE APPOINTED ON RE- QUEST OF THE BOARD OF TRUSTEES, AUGUST 10, 1937, TO FORMULATE A STATEMENT CONCERNING THE POLICIES AND FUTURE OF THE MARINE BIO- LOGICAL LABORATORY I. INTRODUCTION By way of introduction, it is important to remind ourselves of the aims of the founders of the Marine Biological Laboratory. For this purpose a series of quotations follows. It is not the intention to present a history in any detail because it will be found that the original state- ments of policies and aims have been carefully observed during the entire history of the Laboratory for the fifty years of its existence. As the first director early remarked, " These policies should be the germ of an indefinite future development " ; and this has been the case. In the First Annual Report of the Marine Biological Laboratory for the year 1888, the Trustees made the following statements : "Foundation. The Marine Biological Laboratory is an outgrowth of a sea-side laboratory maintained at Annisquam, Mass., from 1880 to 1886, by the Women's Education Association of Boston, in cooperation with the Boston Society of Natural History. In 1886, efforts were made by the Association to place the Laboratory on an independent and broader foundation. A circular letter was addressed to many of the leading biologists of the country, reciting what had been already done at Annisquam, and asking for cooperation and counsel. The replies received were most encouraging, testifying to a general and hearty approval of the enterprise, and promising cooperation and support." (P. 7.) " At the first meeting held by this committee, its members showed by votes that it was their desire to found a laboratory that should give opportunity for original research as well as for instruction, and soon after appointed the following TRUSTEES Prof. William G. Farlow, Prof. Charles S. Minot, Miss Florence M. Gushing, Miss Susan Minns. Prof. Alpheus Hyatt, Prof. William T. Sedgwick, Mr. Samuel Wells." (P. 8.) The first announcement issued in 1888 contained the following statements : ' The Trustees of the Marine Biological Laboratory earnestly desire to enlist your co-operation in the support of a sea-side laboratory for instruction and investigation in Biology." " It is the desire of the Trustees that the enterprise shall enlist the active support of the universities and colleges of the country. To pre- 16 MARINE BIOLOGICAL LABORATORY vent its becoming a simply local undertaking, they wish to see all who aid in its support by subscribing to investigators' tables share with the other members of the Corporation in the annual election of Trustees. The Trustees will, therefore, invite each institution which holds an investigator's table to name five persons for members of the Corporation during the term of subscription." Dr. Whitman commented on these statements in the Eighth Annual Report, for the year 1895 as follows : " Here we see sketched the elemental basis of our germ-organization mainly potentialities of a theoretical nature, but ' instinct with spirit.' The aim was a permanent biological station ; the function was to be instruction and investigation ; the formative principle relied upon was co-operation." (P. 19.) Whitman himself was the most influential person in determining the policies and aims of the new laboratory. In his first annual report as Director in 1888 he stated his personal viewpoint as follows : " The new Laboratory at Woods Hole is nothing more, and, I trust, nothing less, than a first step towards the establishment of an ideal biological station, organized on a basis broad enough to represent all important features of the several types of laboratories hitherto known in Europe and America. It should be provided eventually with means for sending men to different points of the coast to undertake the investi- gation of subjects of special interest, thus adding to the advantages of a fixed station those of an itinerant laboratory. " The research department should furnish just the elements required for the organization of a thoroughly efficient department of instruction. Other things being equal, the investigator is always the best instructor. The highest grade of instruction in any science can only be furnished by one who is thoroughly imbued with the scientific spirit, and who is actually engaged in original work. Hence the propriety and, I may say, the necessity of linking the function of instruction with that of investigation. The advantages of so doing are not by any means con- fined to one side. Teaching is beneficial to the investigator, and the highest powers of acquisition are never reached where the faculty of imparting is neglected. Teaching is an art twice blest; it blesseth him that gives and him that takes. To limit the work of the Laboratory to teaching would be a most serious mistake; and to exclude teaching would shut out the possibilities of the highest development. The com- bination of the two functions in mutually stimulating relations is a feature of the Laboratory to be strongly commended." (Pp. 16-17.) In his lecture on " Specialization and Organization " (Biological Lec- tures, 1890) he remarked : " Among the ways of bringing together our scattered forces into some- thing like organic union, the most important, and the most urgent at REPORT OF THE DIRECTOR 17 this moment, is that of a national marine biological station. Such an establishment, with a strong endowment, is unquestionably the great desideratum of American biology. There is no other means that would bring together so large a number of the leading naturalists of the coun- try, and at the same time place them in such intimate helpful relations to one another. The larger the number of specialists working together, the more completely is the organized whole represented, and the greater and the more numerous the mutual advantages." (P. 24.) In 1893 he wrote in his lecture on " Work and Aims of the Marine Biological Laboratory" (Biological Lectures, 1893): " To those who by word and example have encouraged cooperation, this record will certainly be gratifying; and perhaps it will be accepted by all as an assurance that good-will and united effort have not been fruitless. For six years the Marine Biological Laboratory has stood for the first and the only cooperative organization in the interest of Marine Biology in America." (P. 236.) The same year he remarked in his article " A Marine Observatory the Prime Need of American Biologists " (Atlantic Monthly, June, 1893, pp. 808-815): 'The Marine Biological Laboratory attaches itself to no single insti- tution, but holds itself rigidly to the impartial function of serving all on the same terms. It depends not upon one faculty for its staff of instructors, but seeks the best men it can find among the higher in- stitutions of the land. The board of trustees is a growing body, every year adding to its number, until it now comprises a very large proportion of the leading biologists of America. The whole policy is national in spirit and scope. The laboratory exists in the interest of biology at large, and not to nurse the prestige of any university or the pride of individual pretension." (P. 811.) " Representative character, devotion to biology at large, independent government, such are the essential elements of a strong and progressive organization." (P. 812.) Again in 1898 he returned to the theme in an article " Some of the Functions and Features of a Biological Station " (Science, N.S., Vol. 7, No. 159, January 14, 1898, pp. 11-12) : " It now remains to briefly sketch the general character and to emphasize some of the leading features to be represented in a biological station. ' The first requisite is capacity for growth in all directions con- sistent with the symmetrical development of biology as a whole. The second requisite is the union of the two functions, research and instruc- tion, in such relations as will best hold the work and the workers in the natural coordination essential to scientific progress and to individual development. It is on this basis that I would construct the ideal and test every practical issue. 18 MARINE BIOLOGICAL LABORATORY " A scheme that excludes all limitations except such as nature pre- scribes is just broad enough to take in the science, and that does not strike me as at all extravagant or even as exceeding by a hair's breadth the essentials. Whoever feels it an advantage to be fettered by self- imposed limitations will part company with us here. If any one is troubled with the question: Of what use is an ideal too large to be realized ? I will answer at once. It is the merit of this ideal that it can be realized just as every sound ideal can be realized, only by gradual growth. An ideal that could be realized all at once would exclude growth and leave nothing to be done but to work on in grooves. That is precisely the danger we are seeking to avoid. " The two fundamental requisites which I have just defined scarcely need any amplification. Their implications, however, are far-reaching, and I may, therefore, point out a little more explicitly what is involved. I have made use of the term ' biological station ' in preference to those in more common use, for the reason that my ideal rejects every artificial limitation that might check growth or force a one-sided development. I have in mind, then, not a station devoted exclusively to zoology, or exclusively to botany, or exclusively to physiology ; not a station limited to the study of marine plants and animals ; not a lacustral station deal- ing only with land and fresh-water faunas and floras ; not a station limited to experimental work, but a genuine biological station, embrac- ing all these important divisions, absolutely free of every artificial restriction. "Now, that is a scheme than can grow just as fast as biology grows, and I am of the opinion that nothing short of it could ever adequately represent a national center of instruction and research in biology. Vast as the scheme is, at least in its possibilities, it is a true germ, all the principal parts of which could be realized in respectable beginnings in a very few years and at no enormous expense. With scarcely anything beyond our hands to work with, we have already succeeded in getting zoology and botany well started at Woods Hole, and physiology is ready to follow." II. FUTURE PLANS AND POLICIES A. The Problem of Expansion vs. Consolidation Since the erection of the " New Laboratory " in 1923, there has been a steady growth in the attendance of investigators, subject to some recession during the depression, but reaching a peak in 1937 which strained our accommodations to the limit during the greater part of the session. The question is therefore forced upon our attention whether we should limit arbitrarily the number of investigators as we have long since done in the case of students in classes. The only alternative would be to increase our accommodations. Decision of this point would affect various policies, and it should therefore receive first consideration, REPORT OF THE DIRECTOR 19 The Committee have given careful attention to the question of ex- pansion and have reached the unanimous conclusion that it would be wise at this time to consolidate and develop our present plant and organization, and to postpone the question of expansion, or of new construction except as noted below under Library and under Instruction. The main reasons for this opinion are two : first, that the problems of housing and adequate care of a considerably larger number of persons would be difficult in the restricted community in which we find ourselves, and second, the need of prudence which rests upon economic uncertain- ties. It is by no means certain that we may not have to face another period of depression before many years, and this should not find us over-expanded. Each of these considerations can, of course, be de- veloped in detail. B. The Principle of Cooperation Whitman spoke of cooperation as the " formative principle " of the Laboratory. It is illustrated in the national scope of the Laboratory and in its fundamental organization and government. The principles involved in nation-wide institutional representation and cooperation, and in comprehensive membership of the Corporation, are so rooted in our practices and have proved so fruitful as to require only emphasis. C. Organization and Government The inter-relations of Trustees and Corporation as given in the By-laws have operated harmoniously and effectively for a long time. Rules concerning nomination and election of Trustees and members of the Corporation by the respective bodies have been formulated as follows : 1. By the Corporation: August 11, 1931. 1) After considering various methods by which those engaged in in- struction might be represented upon the Board of Trustees, it is believed that the following action by the Corporation will be the best means of insuring such representation : 1 The Corporation affirms its position that instruction in course work is a fundamental part of the work of the Laboratory and should be adequately represented upon the Board of Trustees." 2) ' That the Committee of the Corporation for nomination of Trustees consist of five members, of whom not less than two shall be non-Trustee members and not less than two shall be Trustee mem- bers of the Corporation." 20 MARINE BIOLOGICAL LABORATORY 3) " That on or about July first of each year, the Clerk shall send a circular letter to each member of the Corporation giving the names of the Nominating Committee and stating that this committee desires suggestions regarding nomination." 4) " That the Nominating Committee shall post the list of nomina- tions at least one week in advance of the annual meeting of the Corporation." (Memo: The same committee also makes nominations annually for Treasurer and Clerk of the Corporation.) 2. By the Trustees : August 10, 1937. " Proposals for membership in the Corporation shall be made to the Nominating Committee on or before the first Tuesday of August upon a regular form and endorsed by two members of the Corpora- tion. " With the recognition that rigid and completely standardized requirements for membership in the Corporation of the Marine Biological Laboratory are neither practicable nor desirable, it is recommended that future members of the Corporation shall, in general, be selected from among persons who, by engaging in active research at the Marine Biological Laboratory during substantial portions of at least two summers, shall have become acquainted with the work, aims, and peculiar problems of the Laboratory, and who, by papers published over a period of several years shall have demon- strated a capacity for sustained scientific productiveness not less than that required for full membership in such national societies as the American Society of Zoologists, the Botanical Society of America, and the American Physiological Society. " It is further recommended that in doubtful or border-line cases action on applications for membership shall be deferred until a time when, in the opinion of the Nominating Committee then serving, the status of the applicant has become entirely clear." D. Administration In the course of the years we have developed methods of adminis- tration of the various service departments of the Laboratory that have worked well. It should be the function of the Director and Assistant Director to control the operation of such services. Dr. Jacobs' greatly regretted resignation as Director raises very directly the question of the higher administration. The first two Direc- tors of the Laboratory served without salary, and the routine admin- REPORT OF THE DIRECTOR 21 istration was performed by an Assistant Director on pay, at first part time but later on full time. Then Dr. Jacobs performed the services both of Director and Assistant Director on half time and half pay, and the Business Manager became able with experience to take over many of the duties formerly exercised by the Assistant Director. Though this arrangement worked admirably for the period of its duration, experi- ence showed that it is not reasonable to expect a man of the scientific experience and reputation expected of the Director of this Laboratory to endure indefinitely the limitations of scientific activity imposed by such an arrangement. It seems probable that we cannot return to this plan. As soon as possible we should provide for a full-time resident Director or Assistant Director. This would afford continuous super- vision of the business of the Laboratory and in addition would permit this officer to continue his research work under favorable conditions. Such a resident scientist would attract other scientists during the portion of the year when the Laboratory is little used and would thus help to make it an all-year-round institution. E. Research and Instruction Research and instruction have been companion principles since the foundation of the Laboratory as cited in the introduction to this report. In the maintenance of research and instruction side by side throughout its history, the Marine Biological Laboratory has been outstanding, if not strictly unique. We have stood by the principle that it is the business of the Laboratory to help to produce investigators as well as investiga- tion ; and we believe that it can be shown that our courses of instruction have contributed in an important way to this purpose, and, moreover, that they have been an important factor in the improvement of biological instruction and research throughout the country. Although there has been some opinion among members of the Laboratory since the courses ceased to be an important source of income that we would be better off without courses, this opinion has never prevailed. We believe that our problem is in the way of improvement, not elimination, of instruction. The Laboratory has no program of its own in research, except as defined in its name, and it therefore promotes no specific research proj- ects as official undertakings. It operates entirely on the principle of furnishing facilities to competent investigators, and to beginning in- vestigators who are working under qualified direction. No biological subjects are specifically excluded except such as are ruled out by lack of facilities or suitable conditions, as in the case of pathogenic organisms for example. This has been the rule from the foundation of the Labora- 22 MARINE BIOLOGICAL LABORATORY tory, and the range of research has consequently steadily increased with improvement of facilities. Changes of fashion have of course also occurred, and are reflected in the annual reports. The policy has been to interest the strongest biologists and promising young investigators to bring their work to Woods Hole ; and the degree of success of this policy has been the measure of success and influence of the Laboratory. The future of the Laboratory depends upon the continuance of this policy, and the elimination of conditions that tend to restrict its operation, whether these are based on inadequacy of equip- ment, administrative regulations, or community conditions. This is the most important policy of the Laboratory, if one may be allowed to rank essentials, for it ensures leadership and reputation. To supple- ment this policy the attendance of as many promising young investiga- tors as possible should be encouraged. If the number of investigators admitted is to be definitely restricted, and if the tendency towards an increase in numbers continues, it will be necessary to adopt more definite policies concerning admission of in- vestigators than in the past. These should not, however, be of too binding a character, but rather a definition of principles within which the Director will have free scope for the exercise of his best judgment. The established fees for research accommodations should be con- tinued, and paid by the institution represented as far as possible. When this cannot be done it has been a frequent policy, more in the past than at present, to waive fees for distinguished investigators. Such arrange- ments have often been doubly blessed, in giving and in taking. The cooperation by institutions in the expenses of investigation of their representatives has been a strong stabilizing factor in the history of the Laboratory in more ways than one. This plan has never been more effective than at the present time, but it is important constantly to cultivate it. The Committee recommends the continuance of our historical policy of maintaining courses of instruction. These should be contributory to research, and based upon the advantages of marine material, so that they are in no sense duplications of courses that may equally well be offered by universities. Of such courses there are several kinds. As con- tributory to research it is not meant that all necessarily lead directly to research as a final preparatory step, but that they may sometimes fill essential gaps in education for the kind of biological research intended by the individual. Preference for admission to courses should be given to students whose promise or declared intention indicates a professional career in the field of biology. Such students should, and do, derive great profit, not only from the actual instruction, but also from the scientific contacts that they make at Woods Hole. REPORT OF THE DIRECTOR 23 The Trustees should maintain control of courses to see that proper content and principles of admission are preserved. The Executive Committee has for some time held a conference with the heads of courses each year with these purposes in mind. Strict limitation of the numbers admitted to each course should be observed in the future as in the past. It should also be a policy to provide better and more stable laboratory accommodations. F. Buildings, Equipmnt and Grounds The first question is whether our holdings of real estate are adequate for the future. This can be answered substantially in the affirmative. We already have considerable undeveloped harbor frontage ; we now own all the land on the block on which the original buildings of the Laboratory stood ; in the block immediately north there is only one parcel of land on Center Street not now in our possession ; and there is no immediate reason for attempting to complete our ownership of the re- mainder of the block. For residential purposes we still have unsold lots in the Gansett tract, and no subdivision whatever has been made of the 100 acres in the Devil's Lane Tract. The second question concerns the buildings. Here three main needs present themselves. In the first place, additional stack space for the library is needed. At the present rate of growth the stacks will be fully occupied in very few years. It is essential for the work of the Laboratory that this growth should be continued. Additional space can be provided by a wing to the east of the present library. It has been suggested that the present reading room might be utilized for additional stack space and the catalogue room be converted into a reading room with other neces- sary readjustments ; other suggestions for temporary relief have also been offered. But at most only a short postponement would be afforded in such ways. The problem should be faced and estimates secured for building additional stack space. The second main need is to replace the present wooden buildings with a fireproof building of solid construction. The work of the classes and investigators in the wooden buildings is seriously hampered by vibration, and the buildings do not lend themselves readily to modern installations. These buildings range in age from forty to fifty years, and they con- stitute a real fire hazard. This need should also receive the earnest attention of the Trustees. Additional space is also needed for various technical services neces- sitated by the increasing complexity of important kinds of biological research in recent years, and which are not adequately provided for at 24 MARINE BIOLOGICAL LABORATORY present. Among these needs are those for space for autoclaves and sterilizers, which must now be used in rooms occupied by investigators, space for stills, which are now very disadvantageously housed in the boiler room in the basement of the Brick Building, additional shop space, particularly for use by investigators for relatively simple operations which they can carry out themselves, additional space for housing small animals, dehumidified and air-conditioned rooms, additional dark rooms, etc. Doubtless most of these needs could be cared for on the lower floors of the proposed addition for the Library. They ought, in any event, not to be forgotten. Furthermore, since needs of this sort are likely to increase in future years and are less predictable than the growth of the library, ample reserve space should be provided for them. Our waterfront should be improved by landscaping and other ways so as to furnish a dignified frontage and water approach to the Labora- tory. The George M. Gray Museum should have more adequate hous- ing, and there are numerous other desirable small improvements that should be undertaken as soon as possible. It is becoming increasingly important that the Supply Department be enabled to collect material for research from a wider area. To this end there should be a larger motor boat, and it is highly desirable that a resident naturalist be associated with the department who could study ecological conditions from year to year with a view to establishing sources of more abundant and more varied material for research. The standing Committee in the Supply Department should be asked to formulate the aims and policies of the Department. G. Library The Library Committee should be asked to formulate the aims and policies of the library. H. Apparatus Similarly, the Apparatus Committee should be asked to formulate its aims and policies. /. Finances and Fiscal Policies In 1932 the income from our endowment funds was $55,668, rep- resenting a return of 5 per cent on book value. It is now approximately $43,000, representing a return of 3.8 per cent on book value. The decrease in yield has been due partly to the necessity of refunding opera- tions at lower interest rates ; but the most drastic reductions in income have been suffered on the mortgage participations, some of which have been foreclosed, and others have had the interest rate much reduced. REPORT OF THE DIRECTOR 25 The outstanding arrears of income amounted to $18,094 in 1935 but were reduced to $12,775 at the end of 1937. For three years the income has been supported by payment of arrears, a condition that cannot continue indefinitely. With a loss of annual income from endowment amounting to over $12,000 there has been a considerable increase in attendance, which has not been compensated for by increased fees for research space. The cost of most apparatus and materials has recently risen appreciably and is likely to rise still farther. It is also certain that the progress of biological research will continually create new demands for special ap- paratus and equipment which must be met if the Laboratory is to retain its present position in scientific research. As a matter of fact, the budget of the Laboratory has been kept balanced since the beginning of the de- pression only by economies which have considerably handicapped the work of many of our investigators. Furthermore, although necessary upkeep has been maintained, certain desirable repairs to the buildings and equipment have been postponed since the early years of the depres- sion but cannot be deferred much longer. Among the more expensive items that will require attention within the next few years are battery replacement, a new heating system for the Brick Building, repairs of the salt water system, painting and waterproofing of the brick buildings, etc. Reserves should also be built up to cover further depreciation of the buildings and equipment owned by the Laboratory, and to provide for the retirement of the Howes mortgage and for future purchases of property, etc. The problem of sewage disposal which may arise at any time is also likely to involve very considerable expense. It is clear that substantial increase of the endowment of the Labora- tory is necessary if we are to aim to restore the income to its pre- depression value, to provide adequately for the upkeep of the present plant, for the establishment of necessary reserves, and to meet increas- ing costs of operation. As a partial offset to the loss of endowment income since 1931, the dividends of the General Biological Supply House increased from $2,032 in 1931 to $12,700 in 1936. The income from fees of students and in- vestigators cannot be increased much unless considerably higher rates are established, which seems undesirable. The Committee agrees that the most important fiscal policies to pur- sue are first to increase endowment and second to establish cash reserves for depreciation and contingencies. It is fair to point out in the latter connection that cash reserves previously accumulated have been used for purchase of real estate and that considerable sums have also gone each year into capital improvements. The Committee recommends that 26 MARINE BIOLOGICAL LABORATORY additional endowments secured be placed in the same trusts as the present major endowment funds, or in another trust under the same principles. /. Community Arrangements and Responsibilities As our community has grown and assumed a more settled character, community needs have increased. The primitive needs of food and lodging have from the beginning been recognized as an official respon- sibility of the Laboratory ; the present arrangements for low-cost hous- ing do not appear to be entirely adequate for a community of our size. Their administration should be in the hands of the Business Manager subject to control by the superior administrative officers. An advisory committee is not recommended. For those who desire to own their own homes, the Laboratory possesses ample real estate in the Gansett and Devil's Lane tracts, sale- able to members at reasonable rates and terms. The acquisition of these tracts has aided to prevent unreasonable increase of price of village properties. These provisions should be regarded as terminating the direct and exclusive official responsibility of the Laboratory for community pur- poses. While the Laboratory should aid in securing recreational facili- ties, the responsibility for operating them should be in the hands of the community itself. This principle has operated well in the case of the " M. B. L. Club " and the " M. B. L. Tennis Club." The Laboratory has furnished land and buildings, and from time to time has made loans for improvements, and it may yet appear desirable to provide an addition to the building of the M. B. L. Club. But these organizations should operate under their own membership and fees. With the acquisition of the bathing beach the question arises whether the same principles could not be made to operate there. K. Summary of Principal Recommendations 1. That the Marine Biological Laboratory pursue a policy of consolida- tion rather than expansion for the present. 2. That in pursuit of this policy steps be taken to provide the following major improvements : a. Secure additional funds for endowment. /'. Provide additional stack room to accommodate approximately 100,000 additional volumes, together with study cubicles. c. Replace present wooden laboratories by a building, or buildings, of stable fireproof construction, providing an intermediate court to set off the present main building. REPORT OF THE DIRECTOR 27 d. In connection with the library construction provide adequate space for expansion of various technical services as described in II F. e. Make provision for the series of miscellaneous needs enumerated in the body of the report. 3. Maintain the principles of cooperation (II B.), organization and gov- ernment (II C.), administration (II D.), research and instruction, (II E.), that have served so well in the past, as the basis for future development. 4. Additional endowment funds as received should be placed, like the present main endowment funds, in trust. Reserves for depreciation, contingencies, improvements and retirement fund should be set up out of income (II I.). 5. Responsibility for recreational facilities should be placed as far as possible on voluntary organizations within our scientific community (II J.). 2. THE STAFF, 1938 CHARLES PACKARD, Associate Director, Assistant Professor of Zoology, Institute of Cancer Research, Columbia University. ZOOLOGY I. INVESTIGATION GARY N. CALKINS, Professor of Protozoology, Columbia University. E. G. CONKLIN, Professor of Zoology, Princeton University. CASWELL GRAVE, Professor of Zoology, Washington University. H. S. JENNINGS, Professor of Zoology, Johns Hopkins University. FRANK R. LILLIE, Professor of Embryology Emeritus, The University of Chicago. C. E. McCujNG, Professor of Zoology, University of Pennsylvania. S. O. MAST, Professor of Zoology, Johns Hopkins University. T. H. MORGAN, Director of the Biological Laboratory, California Institute of Technology. G. H. PARKER, Professor of Zoology Emeritus, Harvard University. E. B. WILSON, Professor of Zoology Emeritus, Columbia University. LORANDE L. WOODRUFF, Professor of Protozoology, Yale University. II. INSTRUCTION T. H. BISSONNETTE, Professor of Biology, Trinity College. P. S. CROWELL, JR., Instructor in Zoology, Miami University. C. E. HADLEY, Associate Professor of Biology, New Jersey State Teachers College at Montclair. F. R. KILLE, Assistant Professor of Zoology, Swarthmore College. A. M. LUCAS, Associate Professor of Zoology, Iowa State College. S. A. MATTHEWS, Assistant Professor of Biology, Williams College. A. J. WATERMAN, Assistant Professor of Biology, Williams College. 28 MARINE BIOLOGICAL LABORATORY JUNIOR INSTRUCTORS W. F. HAHNERT, Associate Professor of Zoology, Ohio Wesleyan Univer- versity. J. S. RANKIN, Teaching Fellow in Zoology, Amherst College. PROTOZOOLOGY I. INVESTIGATION (See Zoology) II. INSTRUCTION GARY N. CALKINS, Professor of Protozoology, Columbia University. G. W. KIDDER, Assistant Professor of Biology, Brown University. ELIZABETH DRUMTRA HUGHES, Lecturer in Zoology, Barnard College. EMBRYOLOGY I. INVESTIGATION (See Zoology) II. INSTRUCTION W. W. BALLARD, Assistant Professor of Biology and Anatomy, Dartmouth College. HUBERT B. GOODRICH, Professor of Biology, Wesleyan University. VIKTOR HAMBURGER, Assistant Professor of Zoology, Washington Univer- sity. OSCAR SCHOTTE, Assistant Professor of Biology, Amherst College. DOUGLAS M. WHITAKER, Professor of Zoology, Stanford University. PHYSIOLOGY I. INVESTIGATION WILLIAM R. AMBERSON, Professor of Physiology, University of Maryland, School of Medicine. HAROLD C. BRADLEY, Professor of Physiological Chemistry, University of Wisconsin. WALTER E. CARREY, Professor of Physiology, Vanderbilt University Medical School. M. H. JACOBS, Professor of General Physiology, University of Pennsylvania. RALPH S. LILLIE, Professor of General Physiology, The University of Chicago. ALBERT P. MATHEWS, Professor of Biochemistry, University of Cincinnati. II. INSTRUCTION Teaching Staff LAURENCE IRVING, Professor of Biology, Swarthmore College. ROBERT CHAMBERS, Professor of Biology, New York University. REPORT OF THE DIRECTOR 29 J. K. W. FERGUSON, Assistant Professor of Physiology, Ohio State Univer- sity. KENNETH C. FISHER, Assistant Professor of Experimental Biology, Uni- versity of Toronto. C. LADD PROSSER, Assistant Professor of Physiology, Clark University. CARL F. SCHMIDT, Professor of Pharmacology, University of Pennsylvania. F. J. M. SICHEL, Instructor in Physiology, University of Vermont, College of Medicine. BOTANY I. INVESTIGATION C. E. ALLEN, Professor of Botany, University of Wisconsin. S. C. BROOKS, Professor of Zoology, University of California. B. M. DUGGAR, Professor of Physiological and Economic Botany, University of Wisconsin. IVEY F. LEWIS, Professor of Biology, University of Virginia. WM. J. ROBBINS, Professor of Botany, University of Missouri. II. INSTRUCTION WILLIAM RANDOLPH TAYLOR, Professor of Botany, University of Michigan. FRANCIS DROUET, Research Fellow, Yale University. B. F. D. RUNK, Research Fellow, University of Virginia. GENERAL OFFICE F. M. MACNAUGHT, Business Manager. POLLY L. CROWELL, Assistant. EDITH BILLINGS, Secretary. RESEARCH SERVICE AND GENERAL MAINTENANCE SAMUEL E. POND, Technical Mgr. LESTER F. Boss, Technician. G. FAILLA, X-ray Physicist. J. D. GRAHAM, Glassblower. T. E. LARKIN, Superintendent. J. T. SIMONTON, Assistant. W. C. HEMENWAY, Carpenter. ELBERT P. LITTLE, X-ray. LIBRARY PRISCILLA B. MONTGOMERY (Mrs. Thomas H. Montgomery, Jr.), Librarian. DEBORAH LAWRENCE, Secretary. MARY A. ROHAN, S. MABELL THOMBS, Assistants. SUPPLY DEPARTMENT JAMES MC!NNIS, Manager. GEOFFREY LEHY, Collector. MILTON B. GRAY, Collector. WALTER KAHLER, Collector. A. M. HILTON, Collector. F. N. WHITMAN, Collector. A. W. LEATHERS, Shipping Dept. RUTH S. CROWELL, Secretary. GRACE HARMAN, Secretary. 30 MARINE BIOLOGICAL LABORATORY THE GEORGE M. GRAY MUSEUM GEORGE M. GRAY, Curator Emeritus. 3. INVESTIGATORS AND STUDENTS, 1938 Independent Investigators ABRAMOWITZ, A. A., Research Assistant, Harvard University. ADAMS, MARK H., Fellowship in Pneumonia Research, Rockefeller Institute for Medical Research. AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland, School of Medicine. ANDERSON, R. L., Professor of Biology, Johnson C. Smith University. ANDERSON, RUBERT S., Research Associate, Princeton University. ANGERER, C. A., Instructor, University of Pennsylvania. APPEL, FREDERICK W., Associate Professor of Biology, St. John's College. ARMSTRONG, PHILIP B., Professor of Anatomy, University of Alabama, School of Medicine. BALLARD, WILLIAM W., Assistant Professor in Zoology and Anatomy, Dartmouth College. EARTH, LESTER G., Assistant Professor of Zoology, Columbia University. BECK, LYLE V., Research Fellow, University of Pennsylvania, School of Medicine. BEDICHEK, SARAH, Associate Professor of Biology, North Texas Agricultural College. BERNSTEIN, FELIX, Professor of Biometrics, New York University, College of Medicine. BERTALANFFY, LUDWIG VON, Privatdozent an der Universitat Wien, Wien, Germany. BISSONNETTE, T. H., Professor and Head of Biology Department, Trinity College. BLACK, LINDSAY MACLEOD, Assistant, Rockefeller Institute for Medical Research. BOCHE, ROBERT D., Research Assistant, Department of Embryology, Carnegie Institution of Washington. BODIAN, DAVID, National Research Council Fellow in Medicine, University of Michigan. BOETTIGER, EDWARD G., Graduate Student, Harvard University. BOERNSTEIN, WALTER, Honorary Research Fellow, Yale University, School of Medicine. BOTSFORD, E. FRANCES, Assistant Professor of Zoology, Connecticut College. BOZLER, EMIL, Assistant Professor of Physiology, Ohio State University. BRADLEY, HAROLD C., Professor of Physiological Chemistry, University of Wis- consin. BRAMBEL, CHARLES E., Instructor in Zoology, Johns Hopkins University. BRONFENBRENNER, J. J., Professor of Bacteriology and Immunology, Washington University, School of Medicine. BUCK, JOHN B., Research Assistant, Department of Embryology, Carnegie Insti- tution of Washington. BUDINGTON, ROBERT A., Professor of Zoology, Oberlin College. BURTON, ALAN C., Fellow in Medical Physics, Johnson Foundation, University of Pennsylvania. CABLE, RAYMOND M., Assistant Professor of Parasitology, Purdue University. CALKINS, GARY N., Professor of Protozoology, Columbia University. CAROTHERS, E. ELEANOR, Research Associate, University of Iowa. CARPENTER, RUSSELL L., Assistant Professor of Anatomy, College of Physicians and Surgeons, Columbia University. CHAMBERS, ROBERT, Research Professor of Biology, Washington Square College, New York University. REPORT OF THE DIRECTOR 31 CHENEY, RALPH H., Chairman of Biology Department, Professor of Biology, Long Island University. CLAFF, C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts. CLARK, ELEANOR L., Department of Anatomy, University of Pennsylvania, School of Medicine. CLARK, ELIOT R., Professor of Anatomy, University of Pennsylvania, School of Medicine. CLAUDE, ALBERT, Associate, Rockefeller Institute for Medical Research. CLOWES, G. H. A., Director of Research, Lilly Research Laboratories. COLE, ELBERT C., Professor of Biology, Williams College. COLE, KENNETH S., Associate Professor of Physiology, College of Physicians and Surgeons, Columbia University. COLWIN, ARTHUR L., Research Fellow, Osborn Zoological Laboratory, Yale University. COMMONER, BARRY, Graduate Student, Harvard University. CONKLIN, EDWIN G., Professor Emeritus of Biology, Princeton University. COOPER, KENNETH W., Lydig Fellow, Columbia University. COPELAND, D. EUGENE, Assistant, Harvard University. COPELAND, MANTON, Professor of Biology, Bowdoin College. CORSON, SAMUEL A., Instructor, Cell Physiology, Division of General Education, Washington Square College, New York University. COSTELLO, DONALD P., Assistant Professor of Zoology, University of North Carolina. COWLES, RHEINART P., Professor of Zoology, Johns Hopkins University. Cox, EDWARD H., Professor of Chemistry, Swarthmore College. CROASDALE, HANNAH T., Technical Assistant in Zoology, Dartmouth College. CROUSE, HELEN V., Research Assistant, Carnegie Institution of Washington. CROWELL, PRINCE S., JR., Instructor in Zoology, Miami University. CURTIS, W. C., Professor of Zoology, University of Missouri. DENNY, MARTHA, Instructor, Connecticut College. DILLER, IRENE COREY, Research Associate in Zoology, University of Pennsylvania. DILLER, WILLIAM F., Assistant Professor of Zoology, Dartmouth College. DROUET, FRANCIS, Theresa Seessel Research Fellow, Yale University. DURYEE, WILLIAM R., Research Associate in Biology, Washington Square College, New York University. ELFTMAN, HERBERT, Assistant Professor of Zoology, Columbia University. ELWYN, ADOLPH, Associate Professor of Neurology, College of Physicians and Surgeons, Columbia University. FAILLA, G., Physicist, Memorial Hospital, New York City. FENNELL, RICHARD A., Instructor in Zoology, Michigan State College. FERGUSON, J. K. W., Assistant Professor of Physiology, Ohio State University. FISHER, KENNETH C., Assistant Professor of Experimental Biology, University of Toronto. FLORKIN, MARCEL, Professor of Biochemistry, University of Liege, Belgium. FORBES, HENRY S., Associate in Neuropathology. Harvard Medical School. FRIES, E. F., Assistant Professor, College of the City of New York. FRISCH, JOHN A., Professor of Biology, Canisius College. FRY, HENRY J., Visiting Investigator, Cornell University Medical College. FURTH, JACOB, Assistant Professor of Pathology, Cornell University Medical College. CARREY, WALTER E., Professor of Physiology, Vanderbilt University, School of Medicine. GELDARD, FRANK A., Professor of Psychology, University of Virginia. GILMAN, MR. LAUREN C., Laboratory Instructor in Biology, Johns Hopkins University. GLASER, OTTO, Professor of Biology, Amherst College. MARINE BIOLOGICAL LABORATORY GOODRICH, H. B., Professor of Biology, Wesleyan University. GORDON-KONIGES, HELMUT, Fellow, Rockefeller Foundation. GRABAR, PIERRE, Fellow, Rockefeller Foundation, Chef de Laboratoire a 1'Institut Pasteur, Paris, France. GRANT, RONALD, Lecturer in Physiology, McGill University. GRAVE, CASWELL, Professor of Zoology, Washington University. GRAY, PETER, Lecturer in Vertebrate Embryology, Edinburgh University. GUTHRIE, MARY J., Associate Professor of Zoology, University of Missouri. HADLEY, CHARLES E., Associate Professor of Biology, Montclair State Teachers' College. HAHNERT, WILLIAM F., Associate Professor of Zoology, Ohio Wesleyan Univer- sity. HAMBURGER, VIKTOR, Assistant Professor, Washington University. HARRIS, DANIEL L., Instructor, University of Pennsylvania. HARROLD, C. M., Graduate Assistant, New York University. HARTMAN, FRANK A., Chairman and Professor of Physiology, Ohio State Uni- versity. HARVEY, ETHEL BROWNE, Investigator, Princeton University. HARVEY, E. NEWTON, Professor of Physiology, Princeton University. HEILBRUNN, L. V., Associate Professor of Zoology, University of Pennsylvania. HENSHAW, PAUL S., Biophysicist, Memorial Hospital, New York City. HETZER, H. O., Associate Animal Husbandman, United States Department of Agriculture, Washington, D. C. HICKSON, ANNA KELTCH, Research Chemist, Lilly Research Laboratories. HIESTAND, WILLIAM A., Associate Professor of Physiology, Purdue University. HILL, EDGAR S., Instructor in Biochemistry, Washington University. HILL, SAMUEL E., Assistant in General Physiology, Rockefeller Institute for Medical Research. HODGE, CHARLES, 4th, Assistant Professor, Temple University. HODGKIN, ALAN L., Demonstrator in Physiology, Cambridge, England. HOPKINS, DWIGHT L., Research Assistant, Johns Hopkins University. HOWE, H. E., Editor, Industrial and Engineering Chemistry, Washington, D. C. HUGHES, ELIZABETH DRUMTRA, Lecturer in Zoology, Barnard College. HUGHES, ROSCOE D., Assistant in Zoology, Columbia University. HUNNINEN, ARNE V., Professor of Biology, Oklahoma City University. HUNTER, GEORGE W., Ill, Assistant Professor of Biology, Wesleyan University. HUNTER, LAURA N., Assistant Professor, Pennsylvania College for Women. HUTCHINGS, Lois M., Teacher of Biology, Weequahic High School, Newark, N. J. IRVING, LAURENCE, Professor of Biology, Swarthmore College. JACOBS, M. H., Professor of General Physiology, University of Pennsylvania. JEFFERS, KATHARINE R., Instructor in Zoology, Duke University. JENKINS, GEORGE B., Professor of Anatomy, George Washington University. JOHLIN, J. M., Associate Professor of Biochemistry, Vanderbilt University, School of Medicine. JONES, E. RUFFIN, JR., Associate Professor, College of William and Mary. JONES, RUTH McCLUNG, Instructor in Botany and Zoology, Swarthmore College. KARADY, STEPHEN, Assistant Professor, Internal Clinic of the Francis Joseph University, Hungary. KIDDER, GEORGE W., Assistant Professor of Biology, Brown University. KIESE, MANFRED, Rockefeller Fellow, Assistant, Pharmacological Institute of the University of Berlin. KILLE, FRANK R., Assistant Professor of Zoology, Swarthmore College. KINDRED, J. E., Associate Professor of Histology and Embryology, University of Virginia. KNOWLTON, FRANK P., Professor of Physiology, Syracuse University, College of Medicine. KOPAC, M. J., Research Associate, Washington Square College, New York University. REPORT OF THE DIRECTOR KORR, IRVIN M., Instructor in Physiology, New York University, College of Medicine. KRAHL, M. E., Research Chemist, Lilly Research Laboratories. KREEZER, GEORGE, Assistant Professor of Psychology, Cornell University. KRIEG, WENDELL J. S., Instructor in Anatomy, New York University, College of Medicine. KUNITZ, MOSES, Associate, Rockefeller Institute for Medical Research. LANCEFIELD, DONALD E., Associate Professor in Biology, Queens College. LEVY, MILTON, Assistant Professor in Chemistry, New York University, College of Medicine. LIEBMANN, EMIL, Fisheries Service of the British Government in the Near East. LILLIE, FRANK R., Professor of Embryology, Emeritus, The University of Chicago. LILLIE, RALPH S., Professor of General Physiology, The University of Chicago. LOEB, LEO, Professor Emeritus of Pathology, Washington University, School of Medicine. LUCAS, ALFRED M., Associate Professor of Zoology, Iowa State College. LUCAS, MIRIAM SCOTT, Iowa State College. LUDWIG, DANIEL, Associate Professor of Biology, New York University. LYNN, W. GARDNER, Instructor, Johns Hopkins University. McCANN, LEWIS P., Graduate Assistant, University of Maryland. McCLUNG, C. E., Director Zoological Laboratory, University of Pennsylvania. McCuRDY, MARY DERRICKSON, Graduate Student, Duke University. McCuRDY, HAROLD G., Research Assistant, Duke University. MACDOUGALL, MARY STUART, Head of Biology Department, Agnes Scott College. McFARLAND, ELSIE LAITY, Instructor in Zoology, Wheaton College. MACLENNAN, RONALD F., Associate Professor of Zoology, State College of Washington. MAGRUDER, SAMUEL R., Assistant in Anatomy, Cornell University Medical College. MALOEUF, N. S. ROYSTON, Honorary Research Fellow, Yale University. MARTIN, W. E., Assistant Professor of Zoology, DePauw University. MAST, S. O., Professor of Zoology in Charge of General Physiology, Johns Hopkins University. MATHEWS, ALBERT P., Andrew Carnegie Professor of Biochemistry, University of Cincinnati. MATTHEWS, SAMUEL A., Assistant Professor of Biology, Williams College. MAYOR, JAMES W., Professor of Biology, Union College. MILLER, JAMES A., Instructor in Anatomy, University of Michigan. MOLTER, JOHN A., Instructor, University of Notre Dame. MORGAN, LILIAN V., Pasadena, California. MORGAN, T. H., Professor of Biology, California Institute of Technology. MORRILL, CHARLES V., Associate Professor of Anatomy, Cornell University Medical College. MULLER, H. J., Institute of Animal Genetics, University of Edinburgh. NAVEZ, ALBERT E., Instructor in Biology, Milton Academy. NEWTON, WILLIAM H., Reader in Physiology, Institute of Physiology, University College, London, England. NONIDEZ, JOSE F., Professor of Anatomy, Cornell University Medical College. NORTHROP, JOHN H., Member, Rockefeller Institute for Medical Research. OBRESHKOVE, VASIL, Professor of Biology, Bard College, Columbia University. O'BRIEN, JOHN P., Johns Hopkins University. OLSON, MAGNUS, Instructor in Zoology, University of Minnesota. ORR, PAUL R., Assistant Professor, Brooklyn College. OSTER, ROBERT H., Assistant Professor, University of Maryland, School of Medicine. OSTERHOUT, W. J. V., Member, Rockefeller Institute for Medical Research. PACKARD, CHARLES, Assistant Professor of Zoology, Institute of Cancer Research, Columbia University. 34 MARINE BIOLOGICAL LABORATORY PARKER, G. H., Professor of Zoology Emeritus, Harvard University. PARMENTER, CHARLES L., Associate Professor, University of Pennsylvania. PARPART, ARTHUR K., Associate Professor, Princeton University. PATRICK, RUTH, Associate Curator of Department of Microscopy, Academy of the Natural Sciences of Philadelphia. PIERCE, MADELENE, Vassar College. PIERSON, BERNICE F., Graduate Student, Johns Hopkins University. PIPKIN, C. A., University of Texas. PLOUGH, HAROLD H., Professor of Biology, Amherst College. POLLISTER, ARTHUR W., Assistant Professor of Zoology, Columbia University. POND, SAMUEL E., Technical Manager, Marine Biological Laboratory. PROSSER, C. LADD, Assistant Professor of Physiology, Clark University. RABINOWITCH, E., Research Associate, University College, London, England. RANKIN, JOHN S., JR., Teaching Fellow in Biology, Amherst College. ROOT, RAYMOND W., Assistant Professor of Biology, College of the City of New York. Rous, PEYTON, Member in Pathology and Bacteriology, Rockefeller Institute for Medical Research. RUGH, ROBERTS, Instructor in Zoology, Hunter College. RUNK, B. F. D., Research Fellow, University of Virginia. RUSSELL, ALICE MARY, Instructor in Zoology, University of Pennsylvania. SABIN, ALBERT B., Associate, Pathology and Bacteriology, Rockefeller Institute for Medical Research. SANDOW, ALEXANDER, Assistant Professor of Biology, Washington Square College, New York University. SASLOW, GEORGE, Instructor in Physiology, Harvard School of Public Health. SAYLES, LEONARD P., Assistant Professor of Biology, College of the City of New York. SCHAEFFER, ASA A., Professor of Biology, Temple University. SCHECHTER, VICTOR, Instructor, College of the City of New York. SCHMIDT, CARL F., Professor of Pharmacology, University of Pennsylvania. SCHMIDT, IDA GENTHER, Assistant Professor of Anatomy, University of Cincin- nati, College of Medicine. SCHMIDT, L. H., Research Fellow, Christ Hospital and University of Cincinnati, College of Medicine. SCHOTTE, OSCAR E., Associate Professor of Biology, Amherst College. SCOTT, ALLAN C., Assistant Professor of Biology, Union College. SCOTT, SISTER FLORENCE MARIE, Professor of Zoology, Seton Hill College. SHAW, MYRTLE, Senior Bacteriologist, New York State Department of Health. SICHEL, ELSA KEIL, Assistant Professor of Zoology, Rutgers University. SICHEL, F. J. M., Instructor in Physiology, University of Vermont, College of Medicine. SLIFER, ELEANOR H., Assistant Professor, State University of Iowa. SMITH, DIETRICH C., Associate Professor of Physiology, University of Maryland, School of Medicine. SMITH, JAY A., Instructor in Biology, Johns Hopkins University. SMITH, MARSHALL E., Student, Johns Hopkins University, Medical School. SOLBERG, ARCHIE N., Instructor in Biology, University of Toledo. SOUTHWICK, MILDRED D., Ecologist, Department of Botany, Vassar College. SPEIDEL, CARL C., Professor of Anatomy, University of Virginia, Medical School. STANLEY, W. M., Associate Member, Rockefeller Institute for Medical Research. STANNARD, J. NEWELL, Instructor in Physiology, University of Rochester, Medical School. STEINBACH, H. BURR, Assistant Professor of Zoology, Columbia University. STEVEN, DAVID M., Magdalen College, Oxford, England. STEINHARDT, JACINTO, Research Fellow, Rockefeller Foundation, Harvard Medi- cal School. REPORT OF THE DIRECTOR STOCKARD, CHARLES R., Professor of Anatomy, Cornell University Medical College. STOKEY, ALMA G., Professor of Botany, Mount Holyoke College. TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan. THORNTON, CHARLES S., Assistant Professor of Biology, Kenyon College. TOWN SEND, GRACE, Professor, Great Falls Normal College. TROMBETTA, VIVIAN V., Assistant in Botany, Barnard College, Columbia University. TURNER, C. L., Professor of Zoology, Northwestern University. TURNER, JOHN P., Assistant Professor of Zoology, University of Minnesota. UHLENHUTH, EDUARD, Professor of Anatomy, University of Maryland, School of Medicine. VANDEBROEK, GEORGES, Assistant in the Laboratory of Embryology and Histology, Faculty of Medicine, University of Ghent, Belgium. VICARI, EMELIA M., Associate in Anatomy, Cornell University Medical College. VISSCHER, J. PAUL, Professor of Biology, Western Reserve University. WALZL, EDWARD M., Instructor, Johns Hopkins University, School of Medicine. WATERMAN, A. J., Assistant Professor of Biology, Williams College. WEISS, PAUL, Associate Professor, The University of Chicago. WENRICH, D. H., Professor of Zoology, University of Pennsylvania. WHITAKER, D. M., Professor of Biology, Stanford University. WHITE, MICHAEL J. D., Lecturer in Zoology, University College, London, England. WHITE, THOMAS N., JR., Assistant Biophysicist, United States Public Health Service, National Institute of Health. WHITING, ANNA R., Guest Research Investigator, University of Pennsylvania. WHITING, P. W., Associate Professor of Zoology, University of Pennsylvania. WICHTERMAN, RALPH, Instructor, Temple University. WIEMAN, H. L., Professor of Zoology, University of Cincinnati. WIERSMA, CORNELIS A. G., Associate Professor of Physiology, California Institute of Technology. WILHELMI, RAYMOND W., Graduate Assistant, New York University. WILLEY, CHARLES H., Assistant Professor of Biology, New York University. WILLIER, BENJAMIN H., Chairman, Division of Biological Sciences, University of Rochester. WILSON, EDMUND B., Professor Emeritus in Residence, Columbia University. WOLF, E. ALFRED, Associate Professor of Biology, University of Pittsburgh. WOLF, OPAL M., Assistant Professor of Biology, Goucher College. WOODRUFF, L. L., Professor of Protozoology, Yale University. YANCEY, P. H., Chairman, Department of Biology, Spring Hill College. YOUNG, ROGER A., Graduate Student, University of Pennsylvania. Beginning Investigators ALGIRE, GLENN H., Weaver Research Fellow in Anatomy, University of Maryland. ARENA, JULIO F. DE LA, Auxiliary Professor of Biology, Universidad de la Habana. BALLENTINE, ROBERT, Graduate Student, Princeton University. BELCHER, JANE C., Graduate Assistant in Zoology, University of Missouri. BELDA, WALTER H., Graduate Student in Zoology, Johns Hopkins University. BISHOP, DAVID W., Instructor, University of Pennsylvania. BLISS, ALFRED F., Laboratory Assistant, Department of Biophysics, Columbia University. BRILL, EDMUND R., Graduate Student in Biology, Harvard University. CASTLE, RUTH M., Assistant in Zoology, Vassar College. CHURNEY, LEON, Instructor in Zoology, University of Pennsylvania. COOPER, RUTH SNYDER, Assistant in Zoology, Columbia University. CORNMAN, IVOR, Teaching Fellow, Washington Square College, New York Uni- versity. CROWELL, VILLA BAILEY, Miami University. 36 MARINE BIOLOGICAL LABORATORY DONNELLON, J. A., Graduate Student, University of Pennsylvania. FERGUSON, FREDERICK P., Undergraduate Assistant, Wesleyan University. FRANK, JOHN A., Medical Student, Yale University. GLANCY, ETHEL, Teaching Fellow, Washington Square College, New York Uni- versity. GOLDIN, ABRAHAM, Graduate Student, Columbia University. GRAVE, CASWELL, II, Assistant, Washington University. GUTTMAN, RITA, Graduate Student in Physiology, College of Physicians and Surgeons, Columbia University. HALL, THOMAS S., Graduate Student, Yale University. HIATT, EDWIN P., Research Fellow, University of Maryland, School of Medicine. HINCHEY, M. CATHERINE, Graduate Student, University of Pennsylvania. HOBSON, LAWRENCE B., Graduate Assistant in Zoology, University of Cincinnati. HOLLINGSWORTH, JOSEPHINE, Graduate Student, University of Pennsylvania. HUTCHINS, Louis W., Graduate Student, Yale University. KRIETE, BERTRAND C., Graduate Assistant in Zoology, University of Cincinnati. LAMBERT, BARBARA, Graduate Assistant in Physiology, Mount Holyoke College. LEVENSON, ALFRED S., Graduate Student, University of Pittsburgh. LIPMAN, HARRY J., Graduate Assistant, University of Pittsburgh. LUDWIG, FRANCIS W., Graduate Student, University of Pennsylvania. MAYO, MERCEDES, Assistant Professor of Biology, Universidad de la Habana. MOORE, ANNA BETTY CLARK, Graduate Student, Columbia University. MOORE, JOHN A., Assistant in Zoology, Columbia University. MULLINS, LORIN J., Graduate Student, University of California. RAMSEY, HELEN J., Purdue University. RAY, D. T., Assistant Professor of Biology, Johnson C. Smith University. ROSE, S. MERYL, Assistant in Zoology, Columbia University. RYAN, FRANCIS J., Assistant in Zoology, Columbia University. SCHENTHAL, JOSEPH E., Weaver Fellow in Anatomy, University of Maryland, School of Medicine. SCHOENBORN, HENRY W., Graduate Assistant, New York University. SCHOEPFLE, G. M., Research Assistant, Princeton University. SHAVER, JOHN R., Museum Assistant, University of Pennsylvania. SILBER, ROBERT H., Assistant and Graduate Student, Washington University. SINGER, MARCUS, Student Worker, University of Pittsburgh. SMITH, AUDREY U., Assistant in Physiology, Vassar College. STEWART, BROOKS, Graduate Student, University of Pennsylvania. VON DACH, HERMAN, Assistant, Ohio State University. WEINBERG, VICTOR, The University of Chicago. WHITE, ELIZABETH C., Student, University of Pennsylvania. WILBUR, KARL M., Harrison Fellow, University of Pennsylvania. WISE, JOHN S., Medical Student, University of Pennsylvania, School of Medicine. ZWILLING, EDGAR, Assistant, Columbia University. Research Assistants ALLEY, ARMINE, Research Assistant, McGill University. ANDERSON, KATHERINE, Research Technician, Vanderbilt University. ARMSTRONG, CHARLES W. J., Demonstrator in Biology, University of Toronto. ARMSTRONG, LOUISE S., Research Assistant, University of Alabama. AURINGER, JACK, Research Assistant, Columbia University. BAKER, LINVILLE A., Lilly Research Laboratories. BECK, NAOMI, Graduate Student, The University of Chicago. BENDER, JOSEPH C., Research Assistant, Swarthmore College. BERNSTEIN, MARIANNE, 325 E. 41st Street, New York City. BERTALANFFY, MARIA M. VON, Universitat Wien, Wien, Germany. REPORT OF THE DIRECTOR 37 BIEN, BETTINA H., Wheaton College. BIRNBAUM, SANFORD M., University Scholar, University of Cincinnati. BIRNBAUM, WILLIAM F., Research Assistant, New York University, College of Medicine. ) BLACK, EDGAR C, Research Associate, Swarthmore College. BOWEN, WILLIAM J., Bruce Fellow, Johns Hopkins University. BROVVNELL, KATHARINE A., Research Assistant, Ohio State University. BURNETT, JACK M., Graduate Student, Washington University. CECIL, SAM, Assistant, Vanderbilt University, School of Medicine. CHAMBERS, EDWARD L., Research Assistant, New York University. COHEN, IRVING, Memorial Hospital, New York City. COSTELLO, HELEN MILLER, University of North Carolina. CRAWFORD, JOHN D., Milton Academy, Milton, Massachusetts. CURTIS, HOWARD J., Associate in Physiology, College of Physicians and Surgeons, Columbia University. DIENES, PRISCILLA, 27 Walker Street, Cambridge, Massachusetts. DOWDING, GRACE L., Research Technician, University of Maryland, School of Medicine. DOWNS, J. HUNTER, Undergraduate, Colgate University. DUGAL, LOUIS-PAUL, Instructor in Biology, University of Montreal. DUMM, MARY E., 13 Sampson Avenue, Madison, New Jersey. DZIEMIAN, ARTHUR J., Graduate Student, Princeton University. EVANS, HIRAM J., Assistant in Biology, Williams College. FINK, HAROLD K., Student, Princeton University. FINKEL, ASHER J., Research Assistant, The University of Chicago. FOSTER, RICHARD, Milton Academy, Milton, Massachusetts. Fox, ERNEST L., Research Assistant, Miami University. FUNKHOUSER, ELIZABETH M. J., Swarthmore College. GETTEMANZ, JOHN F., Laboratory Assistant, Rockefeller Institute for Medical Research. GRAND, C. G., Research Associate, Washington Square College, New York University. HAMDI, TURGUT N., University of Pennsylvania. HATCH, CLEORA, Technician, Cornell University Medical College. HORN, EDWARD C., Assistant, Trinity College. HOWELL, CHARLES D., Professor of Biology, Elizabethtown College. HUTCHENS, JOHN, Lilly Research Laboratories. KEEFE, EUGENE L., Research Assistant, Washington University. KEMP, EMILY J., Instructor in Physiology, University of Maryland, School of Medicine. LEVIN, Louis, Student, University of Cincinnati, College of Medicine. LEWIS, LENA, Research Assistant in Physiology, Ohio State University. LINSCHEID, MARTHA, Research Assistant, Western Reserve University. LYON, RHEA C., Research Technician, University of Maryland, School of Medicine. MCDONALD, MARGARET RITCHIE, Senior Technician, Rockefeller Institute for Medi- cal Research. MARTIN, MARY S., University of Rochester, School of Medicine. MARTIN, ROSEMARY D. C., Assistant, University of Toronto. MELLAND, AMICIA M., Research Worker, Carnegie Institution of Washington. MILFORD, JOHN J., Student, New York University. MUSSER, RUTH E., Student, Goucher College. NAUMANN, RUDOLPH V., Fellow in Physiology, New York University, College of Medicine. NETSKY, MARTIN, Research Assistant, University of Pennsylvania. NORRIS, CHARLES H., Graduate Student, Princeton University. OSBORN, CLINTON M., Research Fellow, Harvard University. 38 MARINE BIOLOGICAL LABORATORY PRATT, DAVID M., Student, Williams College. RAWLES, MARY E., Research Assistant, University of Rochester. SAFFORD, VIRGINIA, Assistant, Swarthmore College. SALZBURG, FREDERICK P., Research Assistant, University of Minnesota. SAWYER, ELIZABETH L., Associate Professor of Biology, Converse College. SCROLL, SAMUEL M., Research Assistant, University of Toledo. SELVERSTONE, BERTRAM, Student, Harvard Medical School. SIMMONS, ERIC L., Research Assistant, Swarthmore College. SISSON, WARREN R., JR., Assistant, Milton Academy. SMITH, CARL C, Iglauer Fellow in Biochemistry, University of Cincinnati. SNEIDER, ELIZABETH, Arnold Biological Fellow, Brown University. SPENCER, JOSEPH M., Research Assistant, College of Physicians and Surgeons, Columbia University. STENGER, ALBERT H., Technician, New York University. STOCKER, GAIL, Research Assistant, University of Pennsylvania. STRICKLAND, J. C., Graduate Instructor, University of Richmond. SUDDATH, E. E., Technician, Washington University. TANERI, BEDIA, Graduate Student, New York University, College of Medicine. THOMPSON, RAYMOND K., Research Assistant, University of Maryland. TUM SUDEN, CAROLINE, Research Fellow in Physiology, Boston University, School of Medicine. WAGNER, CARROLL E., Research Technician, University of Maryland. WIGHTMAN, JOHN C., Assistant in Biology, Brown University. WILSON, JOHN WOODROVV, Graduate Assistant in Zoology, Duke University. YOUNG, SAUL B., Technician, Rockefeller Institute for Medical Research. Students BOTANY BADER, JOAN E., Montclair State Teachers College. BIEN, BETTINA H., Student, Wheaton College. BONNER, JOHN T., Student, Harvard University. FENDER, FLORA S., Preparator, University of Pennsylvania. GRAVES, E. IRENE, Senior Instructor in Biology, Bridgewater State Teachers Col- lege. HOFFMAN, ELIZABETH D., Mount Holyoke College. MARKLE, JANE C., Smith College. POSTEL, FRANCES H., Wellesley College. RUTLEDGE, ALMA W., Graduate Student, Johns Hopkins University. SCHALLEK, WILLIAM B., Harvard University. SIEGEL, MARION T., New Jersey College for Women. WARD, HENRY S., JR., Alabama Polytechnic Institute. EMBRYOLOGY ALLEY, ARMINE, Research Assistant, McGill University. ARMSTRONG, FLORENCE H., Student, Dalhousie University. BERRY, CLYDE, JR., Washington University. BLANCHARD, JOSEPH, Student, Wesleyan University. BOOKHOUT, CAZLYN G., Instructor in Zoology, Duke University. BRUSH, HELEN V., Vassar College. COLLIER, JANE G., Assistant, University of Missouri. COPPOLA, ARMANDO R., Brothers College of Drew University. DOBLER, MARIAN, Goucher College. DRURY, HORACE F., Harvard University. REPORT OF THE DIRECTOR 39 DUNHAM, DONALD W., Assistant in Zoology, Ohio State University. EDDS, MAC VINCENT, JR., Amherst College. FINK, HAROLD K., Graduate Student, Princeton University. FINKEL, ASHER J., Research Assistant, The University of Chicago. HARROLD, CHARLES M., JR., Graduate Assistant, New York University. KLEIN, ETHEL L., University of Rochester. KURTZ, ELIZABETH L., Wilson College. LEWISOHN, MARJORIE G., University of Michigan. MILNE, WALTER S., Graduate Assistant, University of Missouri. PHILIPS, FREDERICK S., Graduate Assistant, University of Rochester. ROGICK, MARY D., Professor of Biology, College of New Rochelle. ROGOFF, WILLIAM M., Graduate Student, Yale University. ROTHERMEL, JULIA E., Professor of Biology, Western College. SODERWALL, ARNOLD L., Assistant in Zoology, University of Illinois. SPANGLER, JULIET M., Wheaton College. STABLEFORD, Louis T., Laboratory Assistant, Yale University. STEVENS, FLORENCE F., New Jersey College for Women. TAYLOR, HARRIETT E., Radcliffe College. TERZIAN, ANNETTE V., Mount Holyoke College. TOWLE, HARRIET N., Assistant in Zoology, Wellesley College. WADDILL, SAMUEL F., Washington and Jefferson College. WILLIAMS, JOHN L., Graduate Assistant, New York University. WOODWARD, ARTHUR A., JR., Student, Oberlin College. WORDEN, FREDERIC G., Student, Dartmouth College. PHYSIOLOGY ALBRINK, WILHELM S., Assistant in Biology, Yale University. ALLEN, PAUL J., Graduate Assistant in Botany, University of Rochester. ARMSTRONG, CHARLES W. J., Demonstrator in Biology, University of Toronto. BECK, NAOMI E., The University of Chicago. BLAIR, JOHN H., Graduate Assistant, Wesleyan University. BLISS, ALFRED F., Columbia University. BRISCOE, PRISCILLA M., Graduate Student, Ohio State University. CASEY, MARGARET T., Graduate Assistant in Physiology, Mount Holyoke College. CROWELL, HAMBLIN H., Graduate Assistant, Ohio State University. CURTIS, HOWARD J., Fellow, Rockefeller Foundation. GRAVE, CASWELL, II, Assistant, Washington University. HENSON, MARGARET, Assistant in Physiology, Wellesley College. LEVINE, HARRY PHILIP, Zoology Instructor, University of Vermont. MARTIN, ROSEMARY D. C, Assistant, University of Toronto. MOORE, IMOGENE, Instructor in Zoology, New Jersey College for Women. MULLINS, LORIN J., University of California. O'BRIEN, JOHN P., Johns Hopkins University. OWENS, WILLIAM C., St. John's College. SMITH, AUDREY U., Assistant in Physiology, Vassar College. VON DACH, HERMAN, Assistant in Zoology, Ohio State University. WIEGHARD, CHARLOTTE, 4544 Harris Avenue, St. Louis, Missouri. WILSON, JOHN W., Graduate Assistant in Zoology, Duke University. PROTOZOOLOGY BEVEL, NELL H., Assistant in Zoology, Duke University. BURBANCK, WILLIAM D., Graduate Assistant, The University of Chicago. COLE, ROGER M., Undergraduate Assistant, Massachusetts State College. EWING, WILLIAM H., Fellow in Biology, Washington and Jefferson College. 40 MARINE BIOLOGICAL LABORATORY FINKELSTEIN, NATHANIEL, Johns Hopkins University. HIERHOLZER, CAROLYN ANNE, Instructor in Biology, Adelphia College. KORNBLUM, LUCILE, Student, Columbia University. MAYO, MERCEDES, Assistant Professor of Biology, Universidad de la Habana. WELLS, WAYNE W., Associate Professor of Science, Southern Oregon State Normal. WILKINSON, ELIZABETH J., Student, Columbia University. INVERTEBRATE ZOOLOGY ACOSTA, JOSEFINA, Goucher College. ALEXANDER, ROBERT S., Graduate Assistant, Amherst College. ARNSTEIN, MARGERY, Simmons College. BELDA, WALTER H., Graduate Student, Johns Hopkins University. BIGLER, FRANCES B., Western Reserve University. BROWN, HENRY, Student, College of the City of New York. COONEY, MARILYN R., Student, Smith College. CRANE, TODD, Student, Wilson College. DAVIS, JAMES O., Graduate Assistant in Zoology, University of Missouri. DELISA, DOMINICK A., Student, Union College. DERINGER, MARGARET K., Johns Hopkins University. DOBBELAAR, MARK E., Teacher of Science, Oradell High School. FAHL, HELEN, Student, Oberlin College. FERGUSON, FREDERICK P., Undergraduate Assistant, Wesleyan University. FLEMING, ROBERT S., Science Critic Teacher, East Carolina Teachers College. FRASER, LEMUEL A., Student, American University. GALE, SHIRLEY, Radcliffe College. GRAVES, E. IRENE, Senior Instructor in Biology, Bridgewater State Teachers College. GRIFFITHS, RAYMOND B., Graduate Research Assistant, Princeton University. HAINES, WILLIAM J., Wabash College. HALL, LYDIA R., Graduate Assistant, Mount Holyoke College. HAMANN, CECIL B., Assistant, Purdue University. HARRIS, NELLIE R., Undergraduate Assistant, Montclair State Teachers College. HOAGLAND, MARY, Swarthmore College. JAEGER, LUCENA, Graduate Assistant in Zoology, University of Missouri. JORDON, ELIZABETH L., Barnard College. JOSEPH, SAMUEL, Student Laboratory Assistant, DePauw University. KELLOGG, MARGARET P., Graduate Student, Cornell University. KERRIGAN, SYLVIA, Graduate Assistant, University of Cincinnati. LINSCHEID, ALFRED G., Western Reserve University. LOVE, GENEVIEVE, Pennsylvania College for Women. MCDONALD, BROWN, Laboratory Assistant, DePauw University. MORRISON, PETER R., Swarthmore College. NADLER, EVELYN R., Brooklyn College. PIERSON, MARY E., Graduate Assistant, Mount Holyoke College. REYER, RANDALL W., Cornell University. ROLLASON, HERBERT D., JR., Middlebury College. ROOT, CHARLOTTE C, Student, Mount Holyoke College. RYAN, THOMAS L, Instructor, Boston College. SACKETT, JOHN T., Graduate, University of Pennsylvania. SANDERS, MARY ELIZABETH, Depauw University. SCHAEFFER, BoBB, Graduate Student, Columbia University. SCHNEIDER, MATHILDA E. C., University of Illinois. SHEEHAN, ELEANOR L., Instructor, University of New Hampshire. SMITH, RALPH I., Harvard University. REPORT OF THE DIRECTOR 41 SNEDECOR, JAMES, Student, Iowa State College. SPERRY, ROGER W., Oberlin College. TABER, ELSIE, Instructor in Biology, Lander College. TOWLE, HARRIET N., Assistant in Zoology, Wellesley College. TROWBRIDGE, CAROLYN F., University of Iowa. WARD, HELEN L., Assistant in Biology, Purdue University. WELCH, D'ALTE A., Johns Hopkins University. WELLS, LORNA A., Graduate Assistant, Oberlin College. WILLIAMS, EDITH M., Student, Elmira College. 4. TABULAR VIEW OF ATTENDANCE 1934 1935 1936 1937 1938 INVESTIGATORS Total 323 315 359 391 380 Independent 222 208 226 256 246 Under Instruction 49 56 76 74 53 Research Assistants 52 51 57 61 81 STUDENTS Total 131 130 138 133 132 Zoology 54 55 55 57 54 Protozoology 11 16 17 16 10 Embryology 30 33 34 35 34 Physiology 23 20 22 16 22 Botany 13 6 10 9 12 TOTAL ATTENDANCE 454 445 497 524 512 Less Persons Registered as Both Students and Investigators 15 16 24 13 16 439 429 473 511 496 INSTITUTIONS REPRESENTED Total 131 143 158 165 151 By Investigators 98 111 120 134 125 By Students 75 70 77 79 67 SCHOOLS AND ACADEMIES REPRESENTED By Investigators 1 2 3 4 By Students 5 3 3 2 1 FOREIGN INSTITUTIONS REPRESENTED By Investigators 4 7 9 16 14 By Students 1 1 5 3 5. SUBSCRIBING AND COOPERATING INSTITUTIONS IN 1938 American University Amherst College Barnard College Belgian American Education Founda- tion, Inc. Bowdoin College Brothers College of Drew University Brown University Bryn Mawr College Carnegie Institute of Washington College of Physicians and Surgeons College of William and Mary Columbia University Purdue University Radcliffe College Rockefeller Foundation Rockefeller Institute for Medical Re- search Rutgers University St. John's College Smith College Spring Hill College State University of Iowa Swarthmore College Syracuse University Temple University 42 MARINE BIOLOGICAL LABORATORY Connecticut College Cornell University Medical College Dalhousie University Dartmouth College DePauw University Duke University Elmira College General Education Board Goucher College Harvard University Harvard University Medical School Hunter College Industrial & Engineering Chemistry, of the American Chemical Society Iowa State College Johns Hopkins University Kenyon College Eli Lilly & Company Long Island University Massachusetts State College Memorial Hospital, New York City Mount Holyoke College New York State Department of Health New York University New York University Medical School Northwestern University Oberlin College Pennsylvania College for Women Princeton University Toledo University Tufts College Union College University of Chicago University of Cincinnati University of Illinois University of Maryland Medical School University of Minnesota University of Missouri University of Notre Dame University of Pennsylvania University of Pittsburgh University of Rochester University of Rochester Medical School University of Vermont University of Virginia Vanderbilt University Medical School Vassar College Wabash College Washington University Washington University Medical School Wellesley College Wesleyan University Western Reserve University Wheaton College Williams College Wilson College Yale University 6. EVENING LECTURES, 1938 Tuesday, June 21 DR. E. H. MYERS "Life Cycle of Foraminifera." Friday, July 1 DR. M. H. JACOBS " Blood and Zoological Classification." Friday, July 8 DR. S. O. MAST " The Synthesis of Living Substance, as Exemplified in Chilomonas paramecium." Friday, July 15 DR. G. H. PARKER " The Color Changes in Animals and Neurohumoral Transmission." Wednesday, July 20 DR. ROBERT CHAMBERS AND DR. WILLIAM DURYEE " Micromanipulation Studies on Cells and Nuclei." Friday, July 22 DR. O. E. SCHOTTE " Induction of Embryonic Organs in Regenerates and Neoplasms." Friday, July 29 DR. EDUARD UHLENHUTH "A Quantitative Approach to the Se- cretion Process of the Thyroid." REPORT OF THE DIRECTOR 43 Friday, August 5 DR. ROBERT CHAMBERS " Structural Aspects of Cell Division." Tuesday, August 9 DR. E. G. CONKLIN AND DR. F. R. LILLIE " Informal Memorial of the Fiftieth Anniversary of the Founding of the Marine Biological Laboratory." Friday, August 12 DR. L. G. EARTH " Studies of the Factors Influencing Regeneration." Friday, August 19 MR. COLUMBUS ISELIN " The Influence of Fluctuations in the Major Ocean Currents on the Cli- mate and the Fisheries." Friday, August 26 DR. PETER GRAY " The Possibility of Affecting Develop- mental Patterns by Electrical Means." Thursday, September 1 (Under the joint auspices of the Genetics Society of America and the Marine Biological Laboratory) DR. H. J. MULLER " The Remaking of Chromosomes." 7. SHORTER SCIENTIFIC PAPERS, 1938 Tuesday, July 5 DR. W. H. NEWTON " Endocrine Activity of the Placenta in Mice." DR. J. K. W. FERGUSON AND DR. H. O. HATERIUS " Evidence for Hormonal Control of Uterine Motility by the Hypoph- ysis in the Rabbit." DR. ROBERTS RUGH " Experimental Studies on the Genital System of the Male Anuran." Tuesday, July 12 DR. G. W. KlDDER AND DR. C. A. STUART " The Role of Chromogenic Bacteria in Ciliate Growth." MR. J. A. SMITH " Some Effects of Temperature on the Reproduction of Chilomonas para- mecium." DR. D. L. HOPKINS " Adjustment of the marine Amoeba, Flabellula mira Schaeffer, to changes in the Total Salt Concen- tration of the Outside Medium." MR. C. L. CLAFF " Phenomena of Excystment in Col- poda cucullus." Tuesday, July 19 DR. K. C. FISHER AND MR. R. OHNELL " The Steady State Frequency of the Embryonic Fish Heart at Differ- ent Cyanide Concentrations." 44 MARINE BIOLOGICAL LABORATORY DR. LENA A. LEWIS " Studies on the Refractory State Re- sulting from the Repeated Injec- tions of Adrenal Extract." DR. EMIL BOZLER " Action Potentials of Visceral Smooth Muscles." DR. L. IRVING " Rhythmical Changes in Blood Flow Through Muscles." Tuesday, July 26 DR. B. H. WlLLIER AND DR. MARY E. RAWLES " Skin Transplants between Embryos of Different Breeds of Fowl." DR. ARTHUR COLWIN " Induction by Cauterization in the Amphibian Egg." DR. VIKTOR HAMBURGER " The Innervation of Transplanted Limbs in Chick Embryos." DR. PAUL WEISS " The Effect of Mechanical Stress on Cartilage Differentiated in Vitro." Tuesday, August 2 DR. J. P. VISSCHER " Some Recent Studies on Barnacles." DR. E. R. JONES, JR " Observations on some of the Lower Turbellaria of the Eastern United States." DR. o'A. A. WELCH " Some Problems of Distribution and Variation in the Hawaiian Tree Snail Achatinella." Tuesday, August 16 DR. D. P. COSTELLO " Studies on Fragments of Centrifuged Nereis Eggs." DR. VICTOR SCHECHTER " Calcium and Magnesium in Relation to the Longevity of Egg Cells." DR. J. B. BUCK AND DR. R. D. BOCHE " Some Properties of Living Chromo- somes." DR. A. M. LUCAS " Some Cytological Studies on Virus- Infected Cells." DR. W. R. DURYEE "A Microdissection Study of Am- phibian Chromosomes." Tuesday, August 23 MR. KARL WILBUR " The Relation of the Magnesium Ion to Ultraviolet Stimulation in the Nereis Egg." DR. E. ELEANOR CAROTHERS " Cytological Effects of X-Rays on Grasshopper Embryos." DR. J. FURTH " Quantitative Studies on the Effect of X-Rays on Mammalian Cells, and on the Mode of X-Ray Ac- tion." DR. P. S. HENSHAW " The Effect of X-Rays on Arbacia punctulata Sperm." DR. T. N. WHITE " Recovery of Arbacia Eggs from High Intensity X-Ray Effects." REPORT OF THE DIRECTOR 45 8. GENERAL SCIENTIFIC MEETING, 1938 Tuesday, August 30 Miss A. M. MELLAND " Isolation of Salivary Gland Nuclei." MR. GLENN H. ALGIRE " Cytological Studies on the Living Thyroid of the Salamander." DR. RALPH H. CHENEY " Micro-Structural Changes in Muscle Fibers after Caffeine." DR. CARL C. SPEIDEL " Some Features of Contraction Nodes and Retraction Clots as Observed in Single Fibers of Cardiac and Skeletal Muscle of Both Verte- brates and Invertebrates." DR. MICHAEL J. D. WHITE " The Heteropycnosis of Sex Chromo- somes and its Interpretation in Terms of Spiral Structure." DR. JOHN P. TURNER " Mitochondria and other Inclusions in the Ciliate Tillina canalifera." DR. ROBERT CHAMBERS " Cytoplasmic Inclusions and Matrix of the Arbacia Egg." DR. M. J. KOPAC " The Devaux Effect at Oil-Proto- plasm Interfaces." DR. M. H. JACOBS AND DR. A. K. PARPART " Further Studies on the Permeability of the Erythrocyte to Ammonium Salts." MR. A. J. DziEMIAN AND DR. A. K. PARPART " Permeability and the Lipoid Content of the Erythrocyte." MR. LOUIS-PAUL DUGAL AND DR. LAURENCE IRVING " The Relation of the Shell to An- aerobic Metabolism in Venus mercenaria." DR. ALEXANDER SANDOW AND DR. KENNETH MORITZ " Tension Output of Muscles in Hypo- tonic Solutions." DR. DWIGHT L. HOPKINS " The Mechanism for the Control of the Intake and the Output of Water by the Vacuoles in the Marine Amoeba, Flabellula mira Schaeffer." DR. N. S. R. MALOEUF "On the Kidney of the Crayfish and the Uptake of Chlorid from Fresh Water by this Animal." DR. N. S. R. MALOEUF " The Osmo-regulative Function of the Alimentary Tract of the Earth- worm, and on the Uptake of Chlo- rid from Fresh Water by this Animal." 46 MARINE BIOLOGICAL LABORATORY DR. ETHEL BROWNE HARVEY " Development of Half-Eggs of Chaetopterus Obtained by Cen- trifugal Force." DR. PAUL S. HENSHAW " The Question of Whether the Delay in Cleavage of Arbacia Eggs Pro- duced with X-Rays is Caused by a General Slowing of the Cleavage Process or by a Block at Some Particular Stage." MR. E. L. CHAMBERS AND DR. ROBERT CHAMBERS " The Resistance of Fertilized Arbacia Eggs to Immersion in KC1 and NaCl Solutions." DR. ALBERT E. NAVEZ " Indolphenoloxidase in Arbacia Eggs and the Nadi Reaction." DR. K. C. COLE AND DR. HOWARD J. CURTIS " Electric Impedance of Nerve Dur- ing Activity." DR. FRANK A. GELDARD " The Vibratory Response of the Skin and its Relation to Pressure Sen- sitivity." DR. E. ALFRED WOLF " Reversal of Phototropic Reaction in Daphnia by the Use of Photosensi- tizing Dyes." DR. CARL C. SPEIDEL " Motion Picture Showing Microscopic Changes in Fibers of Cardiac and Skeletal Muscle of Invertebrates and Vertebrates during Contrac- tion, Retraction, and Clotting." DR. W. R. DURYEE "The Action of Direct Currents on the Cell Nucleus." DR. W. R. DURYEE " Hydration and Dehydration of Fol- licle Cell Nuclei." Wednesday, August 31 DR. HERBERT ELFTMAN "The Function of Muscles in Loco- motion." DR. WILLIAM J. BOWEN " The Effects of Copper and of Vana- dium on the Frequency of Divi- sion." DR. SARAH BEDICHEK " Sex Balance in the Progeny of Trip- loid Drosophila." DR. EDUARD UHLENHUTH, MR. JAMES U. THOMPSON AND MR. JOSEPH E. SCHENTHAL " The Antihormone Problems in the Salamander." DR. ROBERTS RUGH "The Effect of the Sex-Stimulating Factor of the Anterior Pituitary Gland on the Testis of the Bull- frog." REPORT OF THE DIRECTOR 47 DR. J. PAUL VISSCHER " Studies on Barnacle Larvae." DR. GRACE TOWNSEND ''The Spawning Reaction of Nereis limbata with Emphasis Upon Chemical Stimulation." DR. GRACE TOWNSEND " Physiological Assays Concerning the Nature of Fertilizin." DR. ELBERT C. COLE "A Study of the Integument of the Squid, During Staining with Methylene Blue." MR. CARL C. SMITH AND MR. Louis LEVIN " The Use of the Clam Heart as a Test Object for Acetylcholine." DR. OSCAR W. RICHARDS AND Miss KATHARINE J. HAWLEY " The Elimination of Molds." DR. S. E. POND, MR. E. P. LITTLE, MR. A. M. SMITH, AND MR. J. D. GRAHAM "A Comparative Study of Water Aspirators." PAPERS READ BY TITLE DR. C. A. ANGERER " The Effect of Electric Current on the Physical Consistency of Sea Urchin Eggs." MR. C. W. J. ARMSTRONG AND DR. K. C. FISHER " The Effect of Sodium Azide on the Frequency of the Embryonic Fundulus Heart." MR. ROBERT BALLANTINE " Reducing Activity of Fertilized and Unfertilized Arbacia Eggs." DR. LUDWIG VON BERTALANFFY ..." Studies on the Mechanism of Growth in Planaria maculata." MRS. RUTH SNYDER COOPER "Probable Absence of a Chromato- phore Activator in Limulus poly- phemus." MR. C. G. GRAND " Intracellular pH Studies on the Ova of Mactra solidissima." DR. W. R. DURYEE " The Action of Fixatives on the Iso- lated Cell Nucleus." DR. ADOLPH ELWYN " The Melanophore-Expanding Ac- tivity of the Ascidian Neural Gland." MR. RICHARD W. FOSTER, MR. JOHN D. CRAWFORD AND DR. ALBERT E. NAVEZ " Cardiac Rhythm in Pecten irra- dians (Lamarck)." DR. STEPHEN KARADY " The Alarm Reaction and Adaptation Syndrome in Lower Vertebrates (Fundulus majalis)." 48 MARINE BIOLOGICAL LABORATORY DR. M. J. KOPAC " Micro-estimation of Protein Adsorp- tion at Oil-Protoplasm Interfaces." DR. M. J. KOPAC AND DR. R. CHAMBERS " Effect of the Vitelline Membrane on Coalescence of Arbacia Eggs with Oil-drops." DR. GEORGE SASLOW "The Osmotic Pressure of Gum Acacia Solutions." DR. A. A. SCHAEFFER " Differences Between Scottish and American Amebas of the Species Chaos diffluens Miiller." DR. VICTOR SCHECHTER " Induction in Griffithsia." DR. VICTOR SCHECHTER " Bacteria in Relation to Longevity of Egg Cells." DR. J. N. STANNARD " The Effect of Sodium Azide on the Respiration of Frog Muscle." DR. A. J. WATERMAN " Respiratory Stimulants and Gastru- lation in Arbacia." DR. RALPH WICHTERMAN "Does Transfer of Pronuclei ever Occur in Conjugation of Para- mecium caudatum ? " DR. E. ALFRED WOLF AND MR. A. S. LEVENSON " Studies in Calcification. IV. A Contribution to the Problem of Skeletal Calcification in the Tele- ost, Fundulus heteroclitus." DR. OPAL M. WOLF " Mitotic Activity of the Islands of Langerhans and Parathyroids of Rats Following Pituitary Extract and Colchicine Injections." DR. OPAL M. WOLF " Oviducts of Pituitary Stimulated Females, Rana pipiens." Miss R. A. YOUNG " The Effects of Roentgen Irradiatior on Cleavage and Early Develop- ment in the Annelid, Chaetopterm pergamentaceus." MR. E. ZWILLING " The Effect of Perisarc Removal on Regeneration in Tubularia crocea." DEMONSTRATIONS Wednesday, August 31 DR. MICHAEL J. D. WHITE " The Spiral Structure of Animal Chromosomes." DR. P. S. HENSHAW " Cellular Abnormalities Produced by X-Rays." DR. K. S. COLE AND H. J. CURTIS " Electrical Impedance Changes in the Squid Giant Axon Following Ex- citation." REPORT OF THE DIRECTOR 49 DR. E. R. CLARK AND MRS. ELEANOR LINTON CLARK ....a) "Marked Macrophages." b) " Arterio-venous Anastomoses as Observed in the Living Mammal." MR. C. H. NORRIS " Method of Studying Elastic Tension of Marine Eggs." MR. G. H. ALGIRE " Apparatus for the Cytological Study of the Thyroid in the Living Sala- mander." DR. J. P. TURNER " Mitochondria and Other Inclusions in the Ciliate Tillina canalifera." MR. A. S. LEVENSON " Microscopic Sections through Head and Trunk Regions of Fundulus heteroclitus, Prepared by the Gomore Silver Nitrate Method for the Study of Calcification." DR. A. K. PARPART AND MR. S. B. YOUNG " A Simple Glass Electrode System." DR. S. E. POND AND MR. E. P. LITTLE " Water Aspirator Tests and Compari- sons." DR. E. C. COLE a) " Methylene Blue Preparations of the Chromatophores of the Squid." b) "A Low Voltage Lamp for Gen- eral Microscopic Use." c) "Methyl Methacrylate as a Mount- ing Medium for Macroscopic Prep- arations." DR. GRACE TOWNSEND " Spawning Reactions of Male Nereis limbata in Response to Gluta- thione." MR. C. C. SMITH AND MR. Louis LEVIN " The Use of the Clam Heart as a Test Object for Acetylcholine." DR. F. J. M. SICHEL AND DR. S. E. POND " Multi Contact Rheotome." DR. ROBERTS RUGH " Urogenital System of the Male Frog Rana pipiens. Injected to Show the Course of Spermatozoa from the Seminiferous Tubules to the Wolffian Ducts." DR. P. S. GALTSOFF " Sex Reversal in Adult Oysters." DR. P. S. GALTSOFF " Method of Measuring and Recording the Rate of Flow of Water Through the Gills of the Oyster." DR. P. S. GALTSOFF AND MR. GEORGE MISHTOWT " Respiration of the Oyster." 50 MARINE BIOLOGICAL LABORATORY 9. MEMBERS OF THE CORPORATION 1. LIFE MEMBERS ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France. ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Maryland. BILLINGS, MR. R. C, 66 Franklin St., Boston, Massachusetts. CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New Jersey. CRANE, MR. C. R., New York City. EVANS, MRS. GLENDOWER, 12 Otis Place, Boston, Massachusetts. FOOT, Miss KATHERINE, Care of Morgan Harjes Cie, Paris, France. GARDINER, MRS. E. G., Woods Hole, Massachusetts. JACKSON, Miss M. C., 88 Marlboro St., Boston, Massachusetts. JACKSON, MR. CHAS. C., 24 Congress St., Boston, Massachusetts. KING, MR. CHAS. A. LEE, MRS. FREDERIC S., 279 Madison Ave., New York City. LEE, PROF. F. S., College of Physicians and Surgeons, New York City. LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland. LOWELL, MR. A. L., 17 Quincy St., Cambridge, Mass. McMuRRiCH, PROF. J. P., Toronto, Canada. MEANS, DR. J. H., 15 Chestnut St., Boston, Mass. MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Mo. MORGAN, MR. J. PIERPONT, JR., Wall and Broad Sts., New York City. MORGAN, PROF. T. H., Director of Biological Laboratory, California Institute of Technology, Pasadena, California. MORGAN, MRS. T. H., Pasadena, California. MORRILL, DR. A. D., Hamilton College, Clinton, N. Y. NOYES, Miss EVA J. PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsyl- vania. SEARS, DR. HENRY F., 86 Beacon St., Boston, Massachusetts. SHEDD, MR. E. A. THORNDIKE, DR. EDWARD L., Teachers College, Columbia University, New York City. TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, N. Y. TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, Illinois. WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, Calif. WILSON, DR. E. B., Columbia University, New York City. 2. REGULAR MEMBERS, 1938 ABRAMOWITZ, DR. ALEXANDER A., Biological Laboratories, Harvard University, Cambridge, Massachusetts. REPORT OF THE DIRECTOR 51 ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts. ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia, Pennsylvania. ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New York. ALLEE, DR. W. C, The University of Chicago, Chicago, Illinois. ALLYN, DR. HARRIET M., Mount Holyoke College, South Hadley, Massachusetts. AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland, School of Medicine, Lombard and Greene Streets, Baltimore, Maryland. ANDERSON, DR. E. G., California Institute of Technology, Pasadena, California. ANDERSON, DR. RUBERT S., Guyot Hall, Princeton University, Prince- ton, New Jersey. ARMSTRONG, DR. PHILIP B., Syracuse University, Syracuse, New York. AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts. BAITSELL, DR. GEORGE A., Yale University, New Haven, Connecticut, BAKER, DR. H. B., University of Pennsylvania, Philadelphia, Pennsyl- vania. BALDWIN, DR. F. M., University of Southern California, Los Angeles, California. BALL, DR. ERIC G., Johns Hopkins Medical School, Baltimore, Mary- land. BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hamp- shire. BARD, PROF. PHILIP, Johns Hopkins Medical School, Baltimore, Mary- land. BARRON, DR. E. S. GUZMAN, Department of Medicine, The Univer- sity of Chicago, Chicago, Illinois. EARTH, DR. L. G., Department of Zoology, Columbia University, New York City. BEADLE, DR. G. W., School of Biological Sciences, Stanford Univer- sity, California. BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York. BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana. BENNITT, DR. RUDOLF, University of Missouri, Columbia, Missouri. BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge, Massachusetts. BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cam- bridge, Massachusetts. 52 MARINE BIOLOGICAL LABORATORY BINFORD, PROF. RAYMOND, Guilford College, Guilford College, North Carolina. BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut. BLANCHARD, PROF. KENNETH C, Washington Square College, New York University, New York City. BODINE, DR. J. H., Department of Zoology, State University of Iowa, Iowa City, Iowa. BORING, DR. ALICE M., Yenching University, Peking, China. BOZLER, DR. EMIL, Ohio State University, Columbus, Ohio. BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wiscon- sin. BRIDGES, DR. CALVIN B., California Institute of Technology, Pasadena, California. BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, Wash- ington University Medical School, St. Louis, Missouri. BRONK, DR. D. W., University of Pennsylvania, Philadelphia, Pennsyl- vania. BROOKS, DR. S. C., University of California, Berkeley, California. BROWN, DR. DUGALD E. S., New York University, College of Medicine, New York City. BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts. BUDINGTON, PROF. R. A., Oberlin College, Oberlin, Ohio. BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia. BUMPUS, PROF. H. C., Duxbury, Massachusetts. BYRNES, DR. ESTHER E., 1803 North Camac Street, Philadelphia, Penn- sylvania. CALKINS, PROF. GARY N., Columbia University, New York City. CALVERT, PROF. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania. CANNAN, PROF. R. K., New York University College of Medicine, 477 First Avenue, New York City. CARLSON, PROF. A. J., Department of Physiology, The University of Chicago, Chicago, Illinois. CAROTHERS, DR. E. ELEANOR, Department of Zoology, State University of Iowa, Iowa City. CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massa- chusetts. CARROLL, PROF. MITCHELL, Franklin and Marshall College, Lancaster, Pennsylvania. CARVER, PROF. GAIL L., Mercer University, Macon, Georgia. CATTELL, DR. MC!VEEN, Cornell University Medical College, 1300 York Avenue, New York City. REPORT OF THE DIRECTOR 53 CATTELL, PROF. J. McKEEN, Garrison-on-Hudson, New York. CATTELL, MR. WARE, Garrison-on-Hudson, New York. CHAMBERS, DR. ROBERT, Washington Square College, New York Uni- versity, Washington Square, New York City. CHENEY, DR. RALPH H., Biology Department, Long Island University, Brooklyn, New York. CHIDESTER, PROF. F. E., Auburndale, Massachusetts. CHILD, PROF. C. M., Jordan Hall, Stanford University, California. CLAFF, MR. C. LLOYD, Department of Biology, Brown University, Providence, Rhode Island. CLARK, PROF. E. R., University of Pennsylvania Medical School, Phila- delphia, Pennsylvania. CLARK, DR. LEONARD B., Union College, Schenectady, New York. CLELAND, PROF. RALPH E., Indiana University, Bloomington, Indiana. CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana. COE, PROF. W. R., Yale University, New Haven, Connecticut. COHN, DR. EDWIN J., 183 Brattle Street, Cambridge, Massachusetts. COLE, DR. ELBERT C., Department of Biology, Williams College, Wil- liamstown, Massachusetts. COLE, DR. KENNETH S., College of Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York City. COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin. COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio. COLTON, PROF. H. S., Box 601, Flagstaff, Arizona. COONFIELD, DR. B. R., Brooklyn College, 80 Willoughby Street, Brook- lyn, New York. COPELAND, PROF. MANTON, Bowdoin College, Brunswick, Maine. COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina. COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Carolina, Chapel Hill, North Carolina. COWDRY, DR. E. V., Washington University, St. Louis, Missouri. CRAMPTON, PROF. H. E., Barnard College, Columbia University, New York City. CRANE, MRS. C. R., Woods Hole, Massachusetts. CROWELL, DR. P. S., JR., Department of Zoology, Miami University, Oxford, Ohio. CURTIS, DR. MAYNIE R., Crocker Laboratory, Columbia University, New York City. CURTIS, PROF. W. C, University of Missouri, Columbia, Missouri. DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan. 54 MARINE BIOLOGICAL LABORATORY DAVIS, DR. DONALD W., College of William and Mary, Williamsburg, Virginia. DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts. DAWSON, DR. J. A., The College of the City of New York, New York City. DEDERER, DR. PAULINE H., Connecticut College, New London, Con- necticut. DILLER, DR. WILLIAM F., Dartmouth College, Hanover, New Hamp- shire. DODDS, PROF. G. S.. Medical School, University of West Virginia, Mor- gantown, West Virginia. DOLLEY, PROF. WILLIAM L., University of Buffalo, Buffalo, New York. DONALDSON, DR. JOHN C., University of Pittsburgh, School of Medi- cine, Pittsburgh, Pennsylvania. DuBois, DR. EUGENE F., Cornell University Medical College, 1300 York Avenue, New York City. DUGGAR, DR. BENJAMIN M., University of Wisconsin, Madison, Wis- consin. DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota. DURYEE, DR. WILLIAM R., Department of Biology, Washington Square College, New York University, New York City. EDWARDS, DR. D. J., Cornell University Medical College, 1300 York Avenue, New York City. ELLIS, DR. F. W., Monson, Massachusetts. FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France. FERGUSON, DR. JAMES K. W., Department of Physiology, Ohio State University, Columbus, Ohio. FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Richmond, Virginia. FISHER, DR. KENNETH C., Department of Biology, University of To- ronto, Toronto, Canada. FLEISHER, DR. MOYER S., School of Medicine, St. Louis University, St. Louis, Missouri. FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Massachusetts. FRY, DR. HENRY J., Cornell University Medical College, 1300 York Avenue, New York City. FURTH, DR. JACOB, Cornell University Medical College, 1300 York Ave- nue, New York City. GAGE, PROF. S. H., Cornell University, Ithaca, New York. GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy Chase, Maryland. REPORT OF THE DIRECTOR 55 CARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee. GATES, PROF. R. RUGGLES, University of London, London, England. GEISER, DR. S. W., Southern Methodist University, Dallas, Texas. GERARD, PROF. R. W., The University of Chicago, Chicago, Illinois. GLASER, PROF. O. C, Amherst College, Amherst, Massachusetts. GOLDFORB, PROF. A. J., College of the City of New York, Convent Ave- nue and 139th Street, New York City. GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut. GOTTSCHALL, DR. GERTRUDE Y., 230 Central Park West, New York City. GRAHAM, DR. J. Y., University of Alabama, University, Alabama. GRAVE, PROF. B. H., DePauw University, Greencastle, Indiana. GRAVE, PROF. CASWELL, Washington University, St. Louis, Missouri. GRAY, PROF. IRVING E., Duke University, Durham, North Carolina. GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City. GUTHRIE, DR. MARY J., University of Missouri, Columbia, Missouri. GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin. HADLEY, DR. CHARLES E., Teachers College, Montclair, New Jersey. HAGUE, DR. FLORENCE, Sweet Briar College, Sweet Briar, Virginia. HALL, PROF. FRANK G., Duke University, Durham, North Carolina. HAMBURGER, DR. VIKTOR, Department of Zoology, Washington Univer- sity, St. Louis, Missouri. HANCE, DR. ROBERT T., University of Pittsburgh, Pittsburgh, Pennsyl- vania. HARGITT, PROF. GEORGE T., Department of Zoology, Duke University, Durham, North Carolina. HARMAN, DR. MARY T., Kansas State Agricultural College, Manhattan, Kansas. HARNLY, DR. MORRIS H., Washington Square College, New York Uni- versity, New York City. HARPER, PROF. R. A., Columbia University, New York City. HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut. HARTLINE, DR. H. KEFFER, University of Pennsylvania, Philadelphia, Pennsylvania. HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey. HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New Jersey. HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachu- setts. 56 MARINE BIOLOGICAL LABORATORY HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie University, Halifax, Nova Scotia. HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts. HAZEN, DR. T. E., Barnard College, Columbia University, New York City. HECHT, DR. SELIG, Columbia University, New York City. HEILBRUNN, DR. L. V., Department of Zoology, University of Penn- sylvania, Philadelphia, Pennsylvania. HENDEE, DR. ESTHER CRISSEY, Russell Sage College, Troy, New York. HENSHAW, DR. PAUL S., Memorial Hospital, 2 West 106th Street, New York City. HESS, PROF. WALTER N., Hamilton College, Clinton, New York. HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio. HILL, DR. SAMUEL E., Department of Biology, Princeton University, Princeton, New Jersey. HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts. HOADLEY, DR. LEIGH, Harvard University, Cambridge, Massachusetts. HOBER, DR. RUDOLF, University of Pennsylvania, Philadelphia, Penn- sylvania. HODGE, DR. CHARLES, IV., Temple University, Department of Zoology, Philadelphia, Pennsylvania. HOGUE, DR. MARY J., 503 N. High Street, West Chester, Pennsylvania. HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Labora- tory of Ind. Hygiene, 25th and E Street, N.W., Washington, D. C. HOOKER, PROF. DAVENPORT, University of Pittsburgh, School of Medi- cine, Department of Anatomy, Pittsburgh, Pennsylvania. HOPKINS, DR. DWIGHT L., Mundelein College, 6363 Sheridan Road, Chicago. Illinois. HOPKINS, DR. HOYT S., New York University, College of Dentistry, New York City. HOWE, DR. H. E., 2702 36th Street, N.W.. Washington, D. C. HOWLAND, DR. RUTH B., Washington Square College, New York Uni- versity, Washington Square East, New York City. HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Virginia. HYMAN, DR. LIBBIE H., 85 West 166th Street, New York City. IRVING, PROF. LAURENCE, Swarthmore College, Swarthmore, Pennsyl- vania. JACKSON, PROF. C. M., University of Minnesota, Minneapolis, Minne- sota. REPORT OF THE DIRECTOR 57 JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsyl- vania, Philadelphia, Pennsylvania. JENKINS, DR. GEORGE B., George Washington University, 1335 M Street, N.W., Washington, D. C. JENNINGS, PROF. H. S., Johns Hopkins University, Baltimore, Mary- land. JEWETT, PROF. J. R., 44 Francis Avenue, Cambridge, Massachusetts. JOHLIN, DR. J. M., Vanderbilt University Medical School, Nashville, Tennessee. JONES, DR. E. RUFFIN, JR., College of William and Mary, Norfolk, Virginia. JUST, PROF. E. E., Howard University, Washington, D. C. KAUFMANN, PROF. B. P., Carnegie Institution, Cold Spring Harbor, Long Island, New York. KEEFE, REV. ANSELM M., St. Norbert College, West Depere, Wisconsin. KEIL, PROF. ELSA M., Zoology Department, New Jersey College for Women, New Brunswick, New Jersey. KIDDER, DR. GEORGE W., Brown University, Providence, Rhode Island. KILLE, DR. FRANK R., Swarthmore College, Swarthmore, Pennsylvania. KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia. KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, 36th Street and Woodland Avenue, Philadelphia, Pennsylvania. KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa. KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York. KNOWER, PROF. H. McE., Woods Hole, Massachusetts. KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York. KOPAC, DR. M. J., Washington Square College, New York University, New York City. KORR, DR. I. M., Department of Physiology, Washington Square Col- lege, New York University, New York City. KRAHL, DR. M. E., Lilly Research Laboratories, Indianapolis, Indiana. KRIEG, DR. WENDELL J. S., New York University, College of Medicine, 477 First Avenue, New York City. LANCEFIELD, DR. D. E., Queens College, Flushing, New York. LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts. LEWIS, PROF. I. F., University of Virginia, Charlottesville, Virginia. LILLIE, PROF. FRANK R., The University of Chicago, Chicago, Illinois. LILLIE, PROF. RALPH S., The University of Chicago, Chicago, Illinois. LINTON, PROF. EDWIN, University of Pennsylvania, Philadelphia, Penn- sylvania. LOEB, PROF. LEO, Washington University Medical School, St. Louis, Missouri. 58 MARINE BIOLOGICAL LABORATORY LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York City. LUCAS, DR. ALFRED M., Zoological Laboratory, Iowa State College, Ames, Iowa. LUCAS, DR. MIRIAM SCOTT, Department of Zoology, Iowa State College, Ames, Iowa. LUCRE, PROF. BALDUIN, University of Pennsylvania, Philadelphia, Penn- sylvania. LUSCOMBE, MR. W. O., Woods Hole, Massachusetts. LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Ave- nue, New York City. LYNCH, DR. RUTH STOCKING, Maryland State Teachers College, Tow- son, Maryland. MACCARDLE, DR. Ross C, School of Medicine, Duke University, Dur- ham, North Carolina. MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia. MACLENNAN, DR. RONALD F., State College of Washington, Pullman, Washington. McCLUNG, PROF. C. E., University of Pennsylvania, Philadelphia, Penn- sylvania. MCGREGOR, DR. J. H., Columbia University, New York City. MACKLIN, DR. CHARLES C., School of Medicine, University of Western Ontario, London, Canada. MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, Boston, Massachusetts. MALONE, PROF. E. F., College of Medicine, University of Cincinnati, Department of Anatomy, Cincinnati, Ohio. MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York. MARSLAND, DR. DOUGLAS A., Washington Square College, New York University, New York City. MARTIN, PROF. E. A., Department of Biology, Brooklyn College, 80 Willoughby Street, Brooklyn, New York. MAST, PROF. S. O., Johns Hopkins University, Baltimore, Maryland. MATHEWS, PROF. A. P., University of Cincinnati, Cincinnati, Ohio. MATTHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Wil- liams College, Williamstown, Massachusetts. MAYOR, PROF. JAMES W., Union College, Schenectady, New York. MAZIA, DR. DANIEL, Department of Zoology, University of Missouri, Columbia, Missouri. MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Penn- sylvania. REPORT OF THE DIRECTOR MEIGS, DR. E. B., Dairy Division Experimental Station, Beltsville, Maryland. MEIGS, MRS. E. B., 1736 M Street, N.W., Washington, D. C. METCALF, PROF. M. M., 51 Annawan Road, Waban, Massachusetts. METZ, PROF. CHARLES W., Johns Hopkins University, Baltimore, Mary- land. MICHAELIS, DR. LEONOR, Rockefeller Institute, 66th Street and York Avenue, New York City. MILLER, DR. J. A., Department of Anatomy, University of Michigan, Ann Arbor, Michigan. MITCHELL, DR. PHILIP H., Brown University, Providence, Rhode Is- land. MOORE, DR. CARL R., The University of Chicago, Chicago, Illinois. MOORE, PROF. J. PERCY, University of Pennsylvania, Philadelphia, Pennsylvania. MORGULIS, DR. SERGIUS, University of Nebraska, Omaha, Nebraska. MORRILL, PROF. C. V., Cornell University Medical College, 1300 York Avenue, New York City. NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Mil- ton, Massachusetts. NEAL, PROF. H. V., Tufts College, Tufts College, Massachusetts. NELSEN, DR. OLIN E., Department of Zoology, University of Pennsyl- vania, Philadelphia, Pennsylvania. NEWMAN, PROF. H. H., The University of Chicago, Chicago, Illinois. NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania. NOBLE, DR. GLADWYN K., American Museum of Natural History, New York City. NONIDEZ, DR. JOSE F., Cornell University Medical College, 1300 York Avenue, New York City. NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New Jersey. OKKELBERG, DR. PETER, Department of Zoology, University of Michi- gan, Ann Arbor, Michigan. OSBURN, PROF. R. C., Ohio State University, Columbia, Ohio. OSTERHOUT, MRS. W. J. V., Rockefeller Institute, 66th Street and York Avenue, New York City. OSTERHOUT, PROF. W. J. V., Rockefeller Institute, 66th Street and York Avenue, New York City. PACKARD, DR. CHARLES, Columbia University, Institute of Cancer Re- search, 168th Street and Broadway, New York City. PAGE, DR. IRVINE H., Lilly Laboratory Clinical Research, Indianapolis City Hospital, Indianapolis, Indiana. 60 MARINE BIOLOGICAL LABORATORY PAPPENHEIMER, DR. A. M., Columbia University, New York City. PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts. PARMENTER, DR. C. L., Department of Zoology, University of Penn- sylvania, Philadelphia, Pennsylvania. PARPART, DR. ARTHUR K* Princeton University, Princeton, New Jersey. PATTEN, DR. BRADLEY M., University of Michigan Medical School, Ann Arbor, Michigan. PAYNE, PROF. F., University of Indiana, Bloomington, Indiana. PEARL, PROF. RAYMOND, Institute for Biological Research, 1901 East Madison Street, Baltimore, Maryland. PEEBLES, PROF. FLORENCE, Chapman College, Los Angeles, California. PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wis- consin. PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts. POLLISTER, DR. A. W., Columbia University, New York City. POND, DR. SAMUEL E., Marine Biological Laboratory, Woods Hole, Massachusetts. PRATT, DR. FREDERICK H., Boston University, School of Medicine, Boston, Massachusetts. PROSSER, DR. C. LADD, Clark University, Worcester, Massachusetts. RAFFEL, DR. DANIEL, Institute of Genetics, Academy of Sciences, Mos- cow, U. S. S. R. RAND, DR. HERBERT W., Harvard University, Cambridge, Massachu- setts. RANKIN, DR. JOHN S., Biology Department, Amherst College, Amherst, Massachusetts. REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massa- chusetts. REESE, PROF. ALBERT M., West Virginia University, Morgantown, West Virginia. DERENYI, DR. GEORGE S., Department of Anatomy, University of Penn- sylvania, Philadelphia, Pennsylvania. REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue, New York City. RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio. RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma. RICHARDS, DR. O. W., Research Department, Spencer Lens Company, 19 Doat Street, Buffalo, New York. RIGGS, LAWRASON, JR., 120 Broadway, New York City. ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio. ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts. REPORT OF THE DIRECTOR 61 ROOT, DR. R. W., Department of Biology, College of the City of New York, Convent Avenue and 139th Street, New York City. ROOT, DR. W. S., College of Physicians and Surgeons, Department of Physiology, 630 West 168th Street, New York City. RUGH, DR. ROBERTS, Department of Zoology, Hunter College, New York City. SASLOW, DR. GEORGE, Harvard School of Public Health, 55 Shattuck Street, Boston, Massachusetts. SAYLES, DR. LEONARD P., Department of Biology, College of the City of New York, 139th Street and Convent Avenue, New York City. SCHAEFFER, DR. ASA A., Biology Department, Temple University, Phila- delphia, Pennsylvania. SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street and Convent Avenue, New York City. SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio. SCHRADER, DR. FRANZ, Department of Zoology, Columbia University, New York City. SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia Uni- versity, New York City. SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Am- herst, Massachusetts. SCHRAMM, PROF. J. R., University of Pennsylvania, Philadelphia, Penn- sylvania. SCOTT, DR. ALLAN C., Union College, Schenectady, New York. SCOTT, DR. ERNEST L., Columbia University, New York City. SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, New Jersey. SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New York. SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and Surgeons, 630 W. 168th Street, New York City. SHAPIRO, DR. HERBERT, Department of Biology, Clark University, Worcester, Massachusetts. SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Michigan. SHUMWAY, DR. WALDO, University of Illinois, Urbana, Illinois. SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont. SIVICKIS, DR. P. B., Pasto Deze 130, Kaunas, Lithuania. SLIFER, DR. ELEANOR H., Department of Zoology, State University of Iowa, Iowa City, Iowa. 62 MARINE BIOLOGICAL LABORATORY SMITH, DR. DIETRICH CONRAD, Department of Physiology, University of Maryland, School of Medicine, Lombard and Greene Streets, Baltimore, Maryland. SNOW, DR. LAETITIA M., Wellesley College, Wellesley, Massachusetts. SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio. SONNEBORN, DR. T. M., Johns Hopkins University, Baltimore, Mary- land. SPEIDEL, DR. CARL C., University of Virginia, University, Virginia. SPENCER, DR. W. P., Department of Biology, College of Wooster, Wooster, Ohio. STABLER, DR. ROBERT M., Department of Zoology, University of Penn- sylvania, Philadelphia. Pennsylvania. STARK, DR. MARY B., New York Homeopathic Medical College and Flower Hospital, New York City. STEINBACH, DR. HENRY BURR, Columbia University, New York City. STERN, DR. CURT, Department of Zoology, University of Rochester, Rochester, New York. STEWART, DR. DOROTHY R., Skidmore College, Saratoga Springs, New York. STOCK ARD, PROF. C. R., Cornell University Medical College, 1300 York Avenue, New York City. STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College, South Hadley, Massachusetts. STRONG, PROF. O. S., College of Physicians and Surgeons, Columbia University, New York City. STUNKARD, DR. HORACE W., New York University, University Heights, New York City. STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasa- dena, California. SUMMERS, DR. FRANCIS MARION, Department of Biology, College of the City of New York, New York City. SUMWALT, DR. MARGARET, Department of Pharmacology, University of Michigan, Ann Arbor, Michigan. SWETT, DR. FRANCIS H., Duke University Medical School, Durham, North Carolina. TAFT, DR. CHARLES H., JR., University of Texas Medical School, Gal- veston, Texas. TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cin- cinnati, Ohio. TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michi- gan. REPORT OF THE DIRECTOR 63 TENNENT, PROF. D. H., Bryn Mawr College, Bryn Mawr, Pennsylvania. TsWiNKEL, DR. L. E., Department of Zoology, Smith College, North- ampton, Massachusetts. TURNER, DR. ABBY, Department of Physiology, Mount Holyoke College, South Hadley, Massachusetts. TURNER, PROF. C. L., Northwestern University, Evanston, Illinois. TYLER, DR. ALBERT, California Institute of Technology, Pasadena, Cali- fornia. UHLENHUTH, DR. EDUARD. University of Maryland, School of Medi- cine, Baltimore, Maryland. UNGER. DR. W. BYERS. Dartmouth College, Hanover, New Hampshire. VISSCHER, DR. J. PAUL, Western Reserve University. Cleveland, Ohio. WAITE, PROF. F. C., Western Reserve University Medical School, Cleve- land, Ohio. WARD, PROF. HENRY B., University of Illinois. Urbana, Illinois. WARREN, DR. HERBERT S., 1405 Greywall Lane, Overbrook Hills, Penn- sylvania. WATERMAN, DR. ALLYN J., Department of Biology, Williams College, Williamstown, Massachusetts. WEISS, DR. PAUL A., Department of Zoology, The University of Chi- cago, Chicago, Illinois. WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Penn- sylvania. WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, North Dakota. WHITAKER, DR. DOUGLAS M., P. O. Box 2514, Stanford University, California. WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania. WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania. WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Nebraska. WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia, Pennsylvania. WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, Ohio. WILLIER, DR. B. H., Department of Zoology, University of Rochester, Rochester, New York. WILSON, PROF. H. V., University of North Carolina, Chapel Hill, North Carolina. WILSON, DR. J. W., Brown University, Providence, Rhode Island. WITSCHI, PROF. EMIL, Department of Zoology, State University of Iowa, Iowa City, Iowa. 64 MARINE BIOLOGICAL LABORATORY WOLF, DR. ERNST, Biological Laboratories, Harvard University, Cam- bridge, Massachusetts. WOODRUFF, PROF. L. L., Yale University, New Haven, Connecticut. WOODWARD, DR. ALVALYN E., Zoology Dej irtment, University of Michigan, Ann Arbor, Michigan. YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medi- cal College, 1300 York Avenue, New York City. YOUNG, DR. B. P., Cornell University, Ithaca, New York. YOUNG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland. ZELENY, DR. CHARLES, University of Illinois, Urbana, Illinois. STUDIES ON THE TREMATODES OF WOODS HOLE II. THE LIFE CYCLE OF STEPHANOSTOMUM TENUE (LINTON) l W. E. MARTIN (From DePauw University and the Marine Biological Laboratory, Woods Hole, Massachusetts) This paper deals with the results of a study of a member of the trematode family Acanthocolpidae obtained during the summers of 1936 and 1938 at the Marine Biological Laboratory at Woods Hole, Mass. No previous experimental work has been done on the life cycles in this family, and consequently the systematic relationships have been in question. This paper throws some light on these prob- lems. The results obtained may be of some economic importance because the adult members of this family are parasitic in marine fishes, several of which are food fishes. A synopsis of this work was given before the American Society of Parasitologists at the 1938 meeting at Richmond, Virginia. HISTORICAL Some of the members of the family Acanthocolpidae were at first assigned to the old pseudogenus, Distomum, and, due to the presence of spines encircling the mouth, were thought to be related to the echinostomes. Nicoll (1915) placed some of the acanthocolpids in the family Allocreadiidae because of the similarity in the arrangement of the reproductive organs in the two groups. Winfield (1929) criticizes Nicoll's classification, stating, "The Stephanochasminae should be ex- cluded (from the Allocreadiidae) because of the Y-shaped excretory bladder, the circle of head spines, and the armed cirrus and vagina." The family name, Acanthocolpidae, was created by Liihe in 1909 to include trematodes whose principal diagnostic characters are: a well- developed prepharynx and pharynx, a very short esophagus, a Y- shaped excretory bladder, the ovary in front of the testes, the uterus between the ovary and the ventral sucker, the cirrus and vagina armed with spines, and the genital opening medially located anterior to the ventral sucker. At present the following seven genera are included in the family: Stephanostomum Looss 1899, Dihemistephanus Looss 1901, Deropristis Odhner 1902, Acanthocolpus Odhner 1905, 1 This work was made possible through the use of the laboratory facilitirs maintained by Purdue University at the Marine Biological Laboratory. 65 66 W. E. MARTIN Acanthopsolus Liihe 1906, Tormopsolus Poche 1925, and Echinostepha- nus Yamaguti 1934. Because of the presence of connections between the excretory bladder and the ceca in the genus Echinostephanus, Yamaguti separated it from the genus Stephanostomum. However, McFarlane (1936) described such connections in Stephanostomum casum (Linton) and indications of them in S. tristephanum. This suggests that a more extensive and intensive study of this character is needed. Reports of observations pertaining to the life cycles of members of this family have appeared from time to time. Lebour (1907) de- scribed a cercaria that developed in rediae in the limpet, Patella vul- gata, which she believed to be the larval form of some member of the genus Stephanostomum. However, this cercaria lacked eyespots, had a long esophagus and a small sac-shaped excretory bladder, all of which were contrary to observations on the adult worms. The same author (1910) described a cercaria from Buccinum undatum which she thought was the larval form of Acanthopsolus lageniformis. This cer- caria possessed eyespots and general characteristics which agreed with the structures of the adult. No experimental work was done to test the validity of her assumption. Some of the cercariae had tails while the majority did not, which, in conjunction with the absence of large glands in the body, was interpreted by Lebour to indicate that no second intermediate host was required. This seems questionable since Linton (1898), Stafford (1904), Liihe (1906), Nicoll and Small (1909), Nicoll (1910), and others have found metacercariae of this family in various species of fishes. Linton (1898) found cysts of Distomum valdeinflatum attached to the peritoneum of Alutera schoepfi and Menidia menidia notata. Stafford (1904) found the cysts of Stephano- chasmus histrix on the fins of Pseudopleuronectes americanus. Liihe (1906) found Stephanochasmus ceylonicus encysted in the subcutaneous tissue of Narcine timlei taken off Dutch Bay, Ceylon. Lebour (1907) reported Stephanochasmus metacercariae, probably 5. baccatus, under the skin of the dab, witch, and long rough dab. Nicoll and Small (1909) discovered the cysts of Stephanochasmus baccatus under the skin of Pleuronectes limanda. They state, " It is not at all improbable that the cercariae of 5. caducus, S. triglae, and 5. baccatus are all to be found encysted in young pleuronectid fishes." Nicoll (1910) re- ported finding cysts of S. baccatus in Drepanopsetta platessoides. Yamaguti (1934) found cysts of Stephanochasmus sp. with 46 collar spines in Lotella physis and Engraulis japonica, S. sp. with 36 collar spines in Argentina kagoshimae, and 5. sp. with 54 collar spines in Bothrocara zesta and Furcimarius sp. He also found Echinostephanus LIFE CYCLE OF STEPHANOSTOMUM TENUE 67 sp. with 40 collar spines encysted in the flesh of Argentina kagoshimae. The same author (1937) reported Stephanochasmus bicoronatus cysts in the body cavity of Acanthogobius hasta and on the gills of Sciaena sp. and Taenioides lacepedi; Echinostephanus hispidis cysts in the flesh of Psendorhombus pentophthalmus and Neopercis sexfasciatus and Tor- mopsolus larvae encysted near the gills of Leiognathus rivulata. MATERIAL AND METHODS The snail, Nassa obsoleta, which serves as the first intermediate host, Menidia menidia notata the second intermediate host, and the puffer, Spheroides maculatus, which serves as the experimental defini- tive host, were all collected in the vicinity of Woods Hole. Naturally infected snails were used as sources of cercariae. Some Menidia and Spheroides were used for experimental feedings while others were re- tained as controls. Living material was used in the study of many of the cercarial structures. Bouin's solution and a saturated aqueous solution of mercuric chloride were used as fixatives. Mayer's paracarmine was used to stain toto mounts, while sectioned material was stained with Ehrlich's hematoxylin. Infected snails, isolated in finger bowls filled with sea water, furnished a plentiful supply of cercariae for the experi- mental infection of Menidia. OBSERVATIONS AND DESCRIPTIONS The life cycle of Stephanostomum tenue involves the production of rediae and cercariae in the digestive gland of the marine snail, Nassa obsoleta, the development of the metacercariae in cysts in the liver of the small fish, Menidia menidia notata, and the maturation of the worm in the intestine of the puffer, Spheroides maculatus. All measurements listed in this paper are expressed in millimeters. The Redia (Figs. 3 and 4) Natural infections of this trematode were found in about A per cent of the several thousand Nassa obsoleta under observation. Some increase in the number of infected snails in the latter part of the sum- mer was noted, which may be correlated with the migratory habits of the hosts of the adult worms. The redia is an elongate, saccular structure with a pharynx and short rhabdocoel gut. The length of the gut, however, varies with age since it is nearly two-thirds the length of the very young redia (Fig. 4). The young redia also exhibits marked motility. The length of the redia varies from 0.14 to 0.66 with an average of about 0.5; the width varies from 0.03 to 0.14 with 68 W. E. MARTIN an average of about 0.10. The pharynx varies from about 0.025 long by 0.028 wide to 0.052 long by 0.029 wide. The number of germ balls and cercariae per redia varies from to 14 for the former and to 5 for the latter. No ambulatory processes were present and no birth pore was observed. The Cercaria (Fig. 1) The cercaria is of the ophthalmoxiphidio type with a simple tail. In swimming the tail is lashed back and forth while the body is held almost straight. In finger bowls of sea water the cercariae swim about for a short time and then settle to the bottom to which they adhere by the tips of their tails. No special glandular bodies were found in the distal region of the tail that might account for this adhesive action. This behavior may be of importance in the completion of the life cycle since the cercariae may become attached to food particles and may be eaten by fishes. The cercaria exhibits a positive response to light. The cuticula of the body is spinous with larger spines on the an- terior end. In addition to the spines there are seven or eight setae projecting from each side of the body. These are irregularly spaced along the entire body length. The oral sucker bears two rows (of 21 each) of alternating large spines about 0.005 long. These spines are easily detached under even slight cover-glass pressure. The body length varies with the degree of contraction from 0.145 to 0.38 with an average of 0.24, while the body width varies from 0.045 to 0.086 with an average of 0.064. The tail averages about 0.183 long by 0.031 wide. The oral sucker averages about 0.031 long by 0.030 wide while the ventral sucker averages about 0.033 long by 0.030 wide. The ventral sucker bears two rows of small papillae with about 65 papillae in each row. Projecting anteriorly above the oral sucker there is a simple spear-shaped stylet about 0.014 long. The mouth EXPLANATION OF PLATE All drawings were made with the aid of a camera lucida. Abbreviations used: CG, cephalic gland; EB, excretory bladder; EG, esophageal gland; ES, eyespot; G, genital anlage; GB, germ ball; GP, genital pore; /, intestine; 0, oral sucker; OS, oral spines; OV, ovary; P, pharynx; PP, prepharynx; S, stylet; T, testes; V, vitellaria; VG, vesicular gland; VS, ventral sucker. FIG. 1. Ventral view of cercaria. FIG. 2. Stylet of cercaria. FIG. 3. Redia with germ balls and cercaria. FIG. 4. Young redia showing elongate intestine. FIG. 5. Metacercaria. FIG. 6. Adult. LIFE CYCLE OF STEPHANOSTOMUM TENIIE J. 5 70 W. E. MARTIN opens into a long narrow prepharynx approximately 0.038 long. The pharynx is subglobular and measures about 0.012 in length and width. The esophagus is extremely short. The rudimentary intes- tine branches just anterior to the ventral sucker and the branches do not extend beyond this organ. Two conspicuous eyespots are located, one on each side of the body, near the oral sucker. Four cephalic glands are located on each side of the body immediately lateral and anterior to the ventral sucker. On each side of the body the ducts from two glands pass anteriad median to the eyespot while the ducts from the other two glands pass anteriad lateral to the eyespot. The ducts of all four glands open to the exterior at the anterior end of the body. Other glands include numerous vesicular glands along the wall of the excretory bladder. The weakly Y-shaped excretory bladder extends almost to the ventral sucker. In some specimens the anterior wall of the bladder has a scalloped appearance. The main collecting ducts arise from the anterior margin of the excretory bladder and pass anteriad to the level of the eyespots where they bend on themselves and pass posteriad to supply both sides of the body. The flame cells are in seven groups of threes, with the first group given off just after the main duct bends posteriorly at the eyespot level. The other groups are given off at intervals along the side of the body. The reproductive system is represented by a mass of deeply stain- ing cells located just posterior to the ventral sucker and partially surrounded by the anterior wall of the excretory bladder. The Metacercaria (Fig. 5) The cercariae are taken into the digestive tract of the second intermediate host, Menidia menidia notata, where they work their way through the intestinal wall and encyst in the liver or mesenteries. No cercariae were observed to penetrate the bodies of the fishes through the skin. The metacercaria increases to several times the size of the cercaria. The 42 collar spines also increase in size until they are approximately 0.050 long. The eyespots and the glandular cells surrounding the excretory bladder undergo disintegration. There is a marked increase in the size of the pharynx. The branches of the intestine develop until they reach to near the posterior end of the body. The metacercaria is held within a rather tough, loose encyst- ment sac. The Adult (Fig. 6) Nearly mature adult worms were obtained by feeding pieces of Menidia liver containing metacercariae to young puffers, Spheroides maculatus. The puffers were examined about two weeks after the LIFE CYCLE OF STEPHANOSTOMUM TENUE 71 initial feeding and the worms were recovered from the intestine. Remnants of the eyespots were still present. The oral spines were the same in number and approximately of the same size as in the metacercaria. The relative proportions of the suckers and the pharynx were about the same as in the metacercaria. Advances in develop- ment over the conditions found in the metacercaria are: the differen- tiated testes and ovary located in the posterior one-third of the body, the reproductive tubes extending from these organs to the genital pore located on the mid-ventral side of the body immediately anterior to the ventral sucker, and the small clusters of vitelline cells extending along the sides of the body from the posterior end of the pharynx to near the posterior end of the body. Complete functional maturity of the reproductive systems had not been attained since no eggs had been produced. The following measurements and description are based on but a few worms so that the range of variation is probably less than would be found with a larger number of individuals. Body length 1.9 to 2.2, width 0.5; oral sucker 0.13 long by 0.18 wide; ventral sucker 0.22 long by 0.25 wide; prepharynx from 0.19 to 0.31 in length by about 0.015 in width near the oral sucker to 0.031 at its widest point near the pharynx; pharynx about 0.22 long by 0.16 to 0.18 in width; esoph- agus 0.04 to 0.07 long; ovary about 0.057 long by 0.03 to 0.038 wide; anterior testis 0.10 to 0.136 long by 0.04 to 0.07 wide, posterior testis 0.09 to 0.14 long by 0.04 to 0.07 wide. Linton (1898) described Distomum tenue from the rectum of the striped bass, Roccus lineatus, collected at Woods Hole. The descrip- tion he gave, with measurements in millimeters, is as follows: oral spines 0.051 long by 0.018 wide at base; esophagus 0.44 long by 0.34 wide (he undoubtedly has used the term esophagus for the pharynx) ; vitellaria voluminous, peripheral in the posterior region; genital aper- ture immediately in front of the ventral sucker; ova not numerous and comparatively large, lying close behind the ventral sucker; ova length 0.088, width 0.044; body length 2.9, width 0.28; diameter of oral sucker 0.26, of ventral sucker 0.38. DISCUSSION AND CONCLUSIONS Most descriptions of the adult members of this family show them to have remnants of eyespots. This may indicate that the family represents a fairly compact, closely related group. When the excre- tory bladder is mentioned at all in the descriptions of species, it is described as Y-shaped. However, in my study of living specimens of Deropristis inflata, a simple tubular or sac-shaped bladder was found. 72 W. E. MARTIN There is very little in the literature on the rest of the excretory system although Pratt (1916) in his description of Stephanochasmus casum showed that the main collecting tubes pass anteriad to near the level of the eyespots without giving off secondary tubes. The arrangement of the reproductive organs in the family Acan- thocolpidae, as was pointed out by Nicoll (1915), is similar to the arrangement of these organs in the family Allocreadiidae. There is also some suggestion of similarity in the excretory systems of these two groups. In addition, the members of both families are primarily parasites of fishes. This suggests a rather close relationship between the two families. However, the elucidation of the life cycles of other genera is needed before a positive statement can be made. The family Acanthocolpidae seems to be cosmopolitan in distri- bution since some of its members have been found in European, Green- land, North American, Japanese, and Ceylonese waters. There has been some confusion in the literature concerning the generic name Stephanostomum. This confusion resulted from Looss' first (1899) naming the genus Stephanostomum and then changing it to Stephanochasmus (1900) because of its similarity to the genus Stephanostoma Danielson and Koren, a genus of Gephyrean worms. SUMMARY It was found that the life cycle of Stephanostomum, tenue involves the development of rediae and cercariae in the marine snail, Nassa obsoleta, the utilization of the small fish, Menidia menidia notata, as the second intermediate host, and the development of the adult worm in the intestine of the puffer, Spheroides maculatus. Although the puffer may serve as the experimental definitive host, the striped bass, Roccus lineatus, is probably a natural one. About .4 per cent of the Nassa obsoleta observed were infected with this parasite. The excretory system of the cercaria is represented by the formula The arrangement of the reproductive organs, some similarity in the excretory systems, and the fact that fishes serve as hosts to the adult worms suggest an affinity of the Acanthocolpidae to the family Allocreadiidae. LITERATURE CITED LEBOUR, MARIE V., 1907. Fish trematodes of the Northumberland coast. North- umberland Sea Fish. Rep. 2367. LEBOUR, MARIE V., 1910. Acanthopsolus lageniformis n. sp., a trematode in the catfish. Northumberland Sea Fish. Comm. Rep. 1909-1910, pp. 29-35. LINTON, E., 1898. Notes on trematode parasites of fishes. Proc. U. S. Nat. Mnx., 20: 507-548. LIFE CYCLE OF STEPHANOSTOMUM TENUE Looss, A., 1899. Weitere Beitrage zur Kenntniss der Trematodenfauna Aegyptens, zugleich Versuch einer natiirlichen Gliederungdes Genus Distomum Retzius. Zool. Jahrb. Abt. Syst., 12: 521-784. Looss, A., 1900. Nachtragliche Bemerkungen zu den Namen der von mir vor- geschlagenen Distomidengattungen. Zool. Anz., 23: 601-608. Looss, A., 1901. Ueber die Fasciolidengenera Stephanochasmus, Acanthochasmus und einigeandere. Centralbl. Bakt. Parasit., 29: 595-606, 628-634, 654-661. LUHE, MAX, 1906. Trematode parasites from the marine fishes of Ceylon. Ceylon Pearl Oyster Fish, and Marine Biol., Pt. 5: 97-108. LUHE, MAX, 1909. Parasitische Plattwiirmer 17. I. Trematodes. In A. Brauer's, Die Siisswasserfauna Deutschlands. MARTIN, W. E., 1938. The life cycle of Stephanostomum tenue (Linton), family Acanthocolpidae. (Abstract.) Jour. ParasitoL, 24 (Supplement): 27. McFARLANE, S. H., 1936. A study of the endoparasitic trematodes from marine fishes of Departure Bay, B.C. Jour. Biol. Bd. Canada, 2: 335-347. NICOLL, WM., 1910. On the entozoa of fishes from the Firth of Clyde. ParasitoL, 3: 322-359. NICOLL, WM., 1915. A list of the trematode parasites of British marine fishes. ParasitoL, 7: 339-378. NICOLL, WM., AND WM. SMALL, 1909. Notes on larval trematodes. Ann. Mag. Nat. Hist. (Ser. 8), 3: 237-246. ODHNER, TH., 1902. Mittheilungen sur Kenntniss der Distomen. II. Drei neue Distomen aus der Gallenblase von Nilfischen. Centralbl. Bakter. Orig. (4) 31: 152-162. ODHNER, TH., 1905. Die Trematoden des arktischen Gebietes. Fauna Arctica, (2) 4: 291-372. POCHE, FRANZ, 1925. Das System der Platoderia. Arch. Naturg., 91: 1-458. PRATT, H. S., 1916. The trematode genus Stephanochasmus Looss in the Gulf ol Mexico. ParasitoL, 8: 229-238. STAFFORD, J., 1904. Trematodes from Canadian fishes. Zool. Anz., 27: 481-495. WINFIELD, G. F., 1929. Plesiocreadium typicum, a new trematode from Amia calva. Jour. ParasitoL, 16: 81-87. YAMAGUTI, S., 1934. Studies on the helminth fauna of Japan. Pt. 2. Jap. Jour. Zool., 5: 249-541. YAMAGUTI, S., 1937. Studies on the helminth fauna of Japan. Pt. 20. Larval trematodes from marine fishes. Jap. Jour. Zool., (3) 7: 491-499. AN HERMAPHRODITE ARBACIA ETHEL BROWNE HARVEY (From the Biological Laboratory, Princeton University, and the Marine Biological Laboratory, Woods Hole, Mass.} Among the many thousands of Arbacia punctulata opened in the course of ten summers at Woods Hole, and many hundreds of Arbacia pustulosa, Spliaer echinus granularis, Paracentrotus lividus and Par echi- nus microtuberculatus opened during several springs at Naples, and many hundreds of Strongylocentrotus drobachiensis, from Maine, I ob- served last summer for the first time an hermaphrodite sea urchin, an Arbacia punctulata, opened on July 4, 1938. One other case of her- maphroditism in Arbacia punctulata has been described by Shapiro (1935); it was found late in the season of 1934 at Woods Hole. His animal had four testes and one ovary. It was fertile inter se, and all the eggs formed fertilization membranes, but the cleavages were de- layed and abnormal. Many blastulae were obtained and 30 per cent gave rise to gastrulae; there was apparently no further development. James Gray (1921) "described a Strongylocentrotus lividus in which three of the gonads were completely female, another almost completely so and the fifth contained both eggs and sperm which were fertile inter se; development of the eggs is not described. Gadd (1907) described a case of hermaphroditism in Strongylocentrotus drobachiensis at the Mourmanschen Biological Station which had four female gonads and one male, but he does not give any details. The above are the only recorded cases of hermaphroditism in sea urchins, and it is indeed a rare phenomenon. The hermaphrodite Arbacia which I found last summer was quite normal in external appearance and of average size. On removing the ventral portion of the shell, as usual in preparing the eggs, the gonads looked normal except that four were red ovaries and the fifth a white testis with sperm oozing out. Photograph 1 is of the five gonads immediately after removal to sea water. Microscopic observation of the living gonads showed that none of them was entirely male or female. The ovaries had a few tubules containing sperm and the testis contained some ova in various stages of development; that is, the gonads were really ovo-testes but predominantly female or male. A portion of a gonad, living and unstained, is shown in Photograph 2; the ovarian tubules are dark (from the red pigment) and the testis tubules are light with scattered pigment spots; a few eggs have been liberated and lie free in the space between the tubules. A stained 74 EXPLANATION OF PLATES -.* ^-t -- JtJ* * vss&Fn PLATE I PHOTOGRAPH 1. Gonads of the hermaphrodite Arbacia, immediately after re- moval from the shell; one testis (white) and four ovaries (black). Note the small piece of testis (white) at edge of the lower right ovary. PHOTOGRAPH 2. Part of a living gonad, showing testis tubules (white with pigment spots) and ovarian tubules (black) containing eggs, as seen under the micro- scope. A few eggs are seen free in the space between the tubules. PHOTOGRAPH 3. Prepared stained section of a gonad predominantly female containing eggs in various stages of maturity. One testis tubule is seen at lower right. PHOTOGRAPH 4. Prepared stained section of a gonad, predominantly mile, containing mostly ripe sperm. One ovarian tubule is seen at lower right. 76 ETHEL BROWNE HARVEY section of a predominantly female gonad is shown in Photograph 3; all the tubules are filled with eggs in various stages of development except the lower right which is mostly testis. Photograph 4 is a section of the predominantly male gonad ; the tubules are all filled with sperm except one at the lower right w r hich contains eggs. Photo- graph 5 is a section of a predominantly female gonad showing greater detail. Sections of normal ovaries and testes are exactly like these except that there is no mixture. As far as I could tell, especially from a study of the living gonads, the eggs and sperm in the hermaphrodite gonad are separate in the small tubules, and do not lie together with- out any partition. The eggs are not fertilized until they have been liberated from the tubules into the sea water, probably because the sperm are not motile until in sea water. As soon as the eggs have poured out from the tubules into the sea water, they are immediately fertilized by the sperm which have poured into the sea water and become active. At any one time, therefore, the fertilized eggs are found in various stages of development. The eggs of the hermaphrodite are perfectly fertile with its own sperm. Normal fertilization membranes are formed, first cleavage takes place normally and at the normal time, and the later cleavages also, and practically all the eggs develop. The only unusual phe- nomenon was the occurrence of giant eggs. These were about 1 per cent of the total and were all of the same size, 96 /u in diameter, giving a volume of 463,000 yu 3 whereas the normal egg has a diameter of 74 /u and a volume of 212,000/u 3 ; the giant eggs are approximately twice the normal volume. The origin of the giant eggs is not known, but they do not arise from fusion of ripe eggs since giant immature eggs also occur. I have found similar giant eggs in other Arbacias but very rarely, and I have also found in another Arbacia normal-sized eggs with giant nuclei. These nuclei measured 25.6 /u in diameter giving a volume of 8,785 /u 3 , whereas the normal nucleus measures 11.5 fj. in diameter, giving a volume of 796 /u 3 ; the giant nuclei are thus about eleven times the normal volume. Eggs in late cleavages (3^ hours, and less, after fertilization at PHOTOGRAPH 5. Prepared stained section of a gonad under higher magnifica- tion to show greater detail. The gonad was predominantly female, but the portion photographed predominantly male. PHOTOGRAPH 6. Self-fertilized eggs 3| hours (21 C.) after opening the animal. Most of the eggs are in late cleavage stages, but some are not so far advanced since they have come from the tubules and been fertilized later than the others. Note the giant eggs, also developing normally. PHOTOGRAPHS 7-9. Normal development of self-fertilized eggs. PHOTOGRAPH 7. Very early pluteus, self-fertilized, 31 hours old. PHOTOGRAPH 8. Pluteus, self-fertilized, 35 hours. PHOTOGRAPH 9. Pluteus, self-fertilized, 48 hours. o 78 ETHEL BROWNE HARVEY 21 C.) are shown in Photograph 6, and one may observe here the giant eggs. The eggs, including the giant ones, develop quite nor- mally and become swimming blastulae at the normal time, 9 hours after fertilization. The blastulae develop into perfectly normal plutei (Photographs 7-9), and these were kept for nine days. The plutei from the giant eggs were indistinguishable from the others which vary greatly in size according to age. The sperm were perfectly normal in fertilizing other eggs as well as the hermaphrodite eggs (98 per cent), and the eggs from the her- maphrodite could be fertilized perfectly well by sperm from another sea urchin. This latter fact was ascertained by putting a small part of an ovary into fresh water for about | minute to kill the sperm on the outside; then the ovary was transferred to sea water. After an hour, only 1 per cent of the eggs shed were fertilized (by a few sperm liberated from the ovotestis after washing). But when the shed eggs were transferred to sea water containing sperm from another animal, 98 per cent were fertilized. The fertilization was therefore made by the sperm of the normal animal. These eggs developed quite nor- mally. The hermaphrodite animal is therefore fertile with other males and females as well as inter se. I think this the first recorded case in which the eggs of an hermaph- rodite sea urchin, self-fertilized, developed absolutely normally to perfect plutei. SUMMARY 1. A rare case of perfect functional hermaphroditism in the sea urchin Arbacia punctulata is described. There were four red gonads predominantly female and one white gonad predominantly male; there were a few tubules of the opposite sex in all the gonads. 2. Fertilization occurred as soon as the sexual products were liber- ated in sea water. 3. The development of the self-fertilized eggs was absolutely nor- mal, in time and morphology, and normal plutei were raised, nine days old. 4. There occurred about 1 per cent of giant eggs; these were twice the normal volume, and they also developed normally. 5. Both the eggs and the sperm also functioned perfectly normally with other normal males and females. LITERATURE CITED GADD, G., 1907. Ein Fall von Hermaphroditismus bei dem Strongylocentrotus droebachiensis O. F. Mull. Zool. Anz., 31: 635. GRAY, J., 1921. Note on true and apparent hermaphroditism in sea urchins. Proc. Camb. Phil. Soc., 20: 481. SHAPIRO, H., 1935. A case of functional hermaphroditism in the sea-urchin, Arbacia punctulata, and an estimate of the sex-ratio. Am. Nat., 69: 286. KARYOKINESIS DURING CLEAVAGE OF THE ZEBRA FISH BRACHYDANIO RERIO EDWARD C. ROOSEN-RUNGE (From the Department of Biology, Brown University} INTRODUCTION The results presented in this paper have been obtained in the course of a comprehensive study on the periodicity of cell division and mitotic rate during development. A discussion of these results, con- fined to observations on teleosts, is a necessary preliminary to the more complete investigation, with history and literature, to be presented later. To obtain a definite picture of the role of cell division in develop- ment, it is necessary to determine not only the number of mitoses which occur at given times and at given places, but also the duration of a single mitosis and the manner in which it proceeds in different stages of development. In spite of many careful investigations on the rate of mitosis, the duration of mitosis at different periods of development has not been sufficiently determined. The most successful method in investigating the role of cell division in development has been that of Richards (1935) and others who tried to determine mitotic activity by means of a mitotic index or the per- centage number of dividing cells. However, it is not possible to tell by this method whether mitoses occur at periodic cycles or are evenly distributed, so that the counting at any time will actually furnish a figure which approximates the average mitotic rate. The present paper deals with the manner and duration of mitosis only during cleavage. The egg of the zebra fish (Brachydanio rerio] , recently described as a favorable laboratory subject (Roosen-Runge, 1938), is especially adapted to this study, because of its rapid development, its trans- parency, and more particularly because the cell nuclei can be easily observed in the living egg. Three lines of investigation will be de- scribed in this paper, namely, (1) the morphology of the living and of the fixed nuclei; (2) the duration of divisions and of mitotic phases; and (3) their reaction to temperature changes. MATERIALS AND METHODS Information concerning the propagation and raising of the eggs of the zebra fish may be found in an earlier paper (Roosen-Runge). For 79 80 EDWARD C. ROOSEN-RUNGE observation of the living egg, a slide with a covering about 1 mm. thick of a mixture of bee's wax and paraffine was used. A hole, the diameter of the egg, was then cut through the layer of wax in order to let the light come through, with a glass ring, 22 mm. wide and 9 mm. high, added to prevent currents from moving the egg. The slide was im- mersed in water in a large dish of about 150 cc. capacity, to insure an abundant oxygen supply. The egg was then oriented in the hole and all observation carried on, with the slide so immersed, by means of a water-immersion lens (Zeiss, X 40), having sufficient depth of focus to make visible the cells inside the cell membrane. Although the use of an oil-immersion lens is also feasible, it is only useful to check up on details which on the whole can be seen just as clearly with the water-immersion lens. The temperature was regulated with an ordinary desk lamp shining from varying distances upon the observation dish. This simple device proved sufficient to keep the temperature constant within the range of half a degree Centigrade, since the amount of water in which the egg was kept, being fairly large, made it possible to control the temperature almost continuously during the period of development. Thus the eggs continued to develop under the microscope without the least sign of disturbance from the beginning of the second to the end of the tenth cleavage, that is, for a period of about three hours. Bouin's solution was used for fixation. The egg membrane and in most cases the yolk were removed after fixation, for it is then quite easy to tear off the membrane from the hardened egg and to remove the brittle yolk. Dioxan or alcohol + benzol was used for dehydra- tion, but the former is the simpler method and, therefore, to be pre- ferred. All sections were stained in Heidenhain's haematoxylin and cut 6 or 8 microns in thickness. MORPHOLOGY OF THE NUCLEI It is impossible to study the nucleus in the living egg before the first cleavage since the delicate structure is then hidden by coarse granules which are whirled up at the base of the cell by the streaming of the protoplasm into the blastodisc. During the first cleavage the streaming still continues, offering some difficulties to the observer. Accurate observation of the nucleus becomes possible only when the cytoplasm clears at the end of the first cleavage. The two nuclei appear as ovals with a longitudinal axis of approximately 18 M- The outlines are fine and smooth. Two or three, sometimes more, very delicate curved lines divide the nucleus into several sections (Fig. 1). The first signs of mitosis are the swelling of the nucleus and the KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 81 appearance of irregularities in its oval shape when tiny indentations can be seen at the poles which appear flattened so that the nucleus assumes a barrel-like shape. Short rays which point toward the center of the nucleus seem to radiate from the depths of the indenta- tions. Very often the nucleus appears to be divided lengthwise into halves by a fine channel which is filled with some substance a shade darker than the nuclear sap. In the living egg the appearance of the indentations marks the beginning of a very rapid disintegration of the nuclear membrane. The whole circumference appears strongly wrinkled and rapidly fades out, together with the partition lines inside of the nucleus. In a short time no traces of nuclear structures are left. By watching very closely, one can for a moment fancy where the nucleus has been, because this area appears somewhat lighter and free from the tiny granules which are a part of the cytoplasm throughout the cell. Before nuclear structures become visible again, the cell almost completes its division. The changes in the cytoplasm and the shape of the cell during mitosis have already been described (Roosen-Runge, 1938). Sometime after the furrow has completely cut through, there ap- pears in the center of each daughter cell a group of tiny dark granules. These granules represent the chromosomes. They swell, become lighter, and finally appear as little circles or vesicles with very distinct outlines. The vesicles go on swelling rapidly until they come into contact with each other, eventually forming one body with a common but irregularly curved contour. The outlines of the individual vesicles remain visible for a time, some of them fading out finally, while others do not disappear until the breakdown of the nuclear membrane in the next prophase. Observations on the living nuclei confirm some of the results ob- tained from sectioned material. The outstanding feature in the karyokinesis of the teleost blastomeres is the formation of chromosomal vesicles during the telophase. These chromosomal vesicles are quite commonly found in early development and are supposed to persist through the interkinetic phase into the prophase. This interpretation has been made very probable by A. Richards (1917) and B. G. Smith (1929) from the study of sections. It can be proved by the study of living nuclei, in which some of the walls of the vesicles can actually be seen to persist in the interphase nucleus. Some of the walls, however, do not remain visible, but this seems to be due to their thinning out and not to their complete disappearance, since the sections also show some partitions, very dark and distinct, while others are delicate and inconspicuous. In many instances the sectioned nuclei can be seen EDWARD C. ROOSEN-RUNGE divided into halves, inside of which the vesicles are visible. The halves are separated by a gap, apparently filled with cytoplasm, which corre- sponds to the observations on the living nuclei. The halves represent the paternal and the maternal parts of the chromosome set, as first described by Moenkhaus (1904) in teleost hybrids, and by many early workers on other forms. How the vesicles arise from the anaphase chromosomes and how the chromosomes are formed from the vesicles in the prophase, cannot be determined accurately from fixed material, nor do observations of the living nuclei solve any of these problems. Richards (1917) concluded that the vesicles are formed by a swelling of the chromosomes so that finally the walls contain the chromatin material and enclose a space "filled in from the fluid portion of protoplasm." Smith (1929), on the other hand, studied the karyokinesis in Cryptobranchus eggs and found that the vesicular membrane was of cytoplasmic origin, de- veloped under the influence of the chromosome within. Pictures like those of Smith certainly cannot be seen in sections of either Fundulus or Brachydanio eggs. The study of living nuclei only confirms the impression that the chromosomes actually swell during the telophase and that the vesicular wall represents the surface of the chromosome rather than a structure formed de novo from the cytoplasm. My own material does not show some of the details as distinctly as they appear (according to Richards) in Fundulus, although the formation of the vesicles and their persistence through the interphase could be clearly seen in the sections as well as in the living egg. Nevertheless, the behavior of the chromosome material still remained puzzling. That the reader may be better able to appreciate its actual appearance, I have used photographs (see plate) rather than drawings, as Richards and others have done. Attempts at drawing present possibly too great a temptation to express a prejudiced interpretation not justified by the actual material. The prophase stage in the karyokinesis of the living blastomere has already been described. The appearance of the nucleus as a whole cor- responds very well with the observations of the sections. Because of the rapidity with which the chromosomes reappear and arrange them- selves, only a few figures in these phases wall be found in material fixed at random. However, by closely watching the living nuclei and taking into account the time necessary for sufficient penetration of the fixing fluid to arrest the mitosis (about half a minute for Bouin's fluid), it is possible to fix material in any desired stage. It can then be seen that the individual chromosomes become clearly visible only immediately before the breakdown of the nuclear membrane. They seem to begin KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 83 6 PLATE I EXPLANATION OF FIGURES FIG. 1. One of the eight blastomeres of a zebra fish egg, living. Nucleus with a few partition lines within, in the center. X 500. FIGS. 2-7. Nuclei, fixed in Bouin's, Heidenhain'shaematoxylin, 6-8 yu. X 1100. The different sizes of the nuclei are due to their belonging to different cleavage stages. FIG. 2. Prophase in the beginning. Vesicles still visible. Indentations at the poles. FIG. 3. Advanced prophase, chromosomes appearing. FIG. 4. Chromosomes forming metaphase plate. Outline of nucleus still visible. FIG. 5. Nuclei from cells after the twelfth cleavage, showing typical spireme formation in the prophase. FIG. 6. Early telophase. Formation of chromosomal vesicles. FIG. 7. Late telophase. Vesicles in contact. Paternal and maternal half of chromosomes apparently separate. 84 EDWARD C. ROOSEN-RUNGE the arrangement into a plate while still inside of the membrane (Fig. 4). This fact has been confirmed by observations on the living eggs of another teleost, Epiplaty chaperi, in which the chromosomes are some- what more easily discernible in life. Directly after the breakdown of the nuclear membrane the chromosomes can be seen arranged in a metaphase plate but very soon afterwards they begin separating. This observation shows that the disappearance of the membrane actually occurs relatively late. Individual chromosomes inside the separate vesicles, as pictured by Richards, could not be found in sections of the zebra fish egg. THE DURATION OF CELL DIVISION The absolute duration of cell division varies tremendously in dif- ferent animals, and in different cells of the same animal, despite the fact that karyokinesis is supposed to occur essentially in the same way in all of them. The duration of mitosis is characteristic for the dif- ferent kinds of cells. It can only be measured accurately through the direct observation of living material. The relative time of mitotic phases has been estimated by using the percentage number of cells active in the different stages, but in many cases, as will be pointed out later, this method is very erroneous. It seems, therefore, that direct observation is the safest method for determining the relative intervals in cell division. The most considerable error in measuring the duration of cell division in life arises from the difficulty in finding any definite point of departure. Neither the beginning of the prophase nor the last stage of the telophase can be defined accurately, so that only a very few events are established sharply enough to serve as marks by which stages may be measured. In the blastomeres of the zebra fish the swelling of the nucleus at the beginning of the prophase, the breakdown of the nucleus, the appearance of the furrow, the completion of the furrow, the first appearance of the chromosomes in the telophase, and finally the completion of the rounded nucleus, furnish seven criteria of very different value. The time of the formation of the furrow, which means the division of the cytoplasm, can only be used indirectly for the determination of karyokinetic stages, although it may serve to subdivide the interval in which nuclear structures cannot be observed at all. The moment when the nucleus seems completely rounded and smoothly outlined is almost impossible to define, and its determination involves a considerable error. The swelling of the nucleus in the early prophase is also difficult to observe, but it is possible to determine its approximate beginning somewhat better with the aid of a micrometer KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 85 eye-piece, the scale of which will permit accurate observation of small changes in size. The reappearance of the chromosomes as tiny gran- ules in the telophase is an event more easily determined. Under favorable conditions it is quite possible to watch the optically empty central area of the cell and to see the chromosomes become visible. I estimate the possible error under optimal conditions to be not more than 30 seconds or 3 per cent of the whole time of cleavage. By far the best mark, because of its rapidity of occurrence, is the breakdown of the nucleus. The nuclear membrane not only disappears in from 15 to 30 seconds, but the onset of this event is foreshadowed by a series of preparatory events, namely, the swelling of the nucleus and the wrinkling of the membrane, which makes it possible to predict the time of breakdown quite accurately. The error in determining the precise time of this occurrence is certainly not greater than 15 seconds, which is about 1.5 per cent of the whole time of cleavage. We have thus found two marks which seem reliable, because their errors can be estimated with considerable accuracy at only 1.5 to 3 per cent of the entire duration of cleavage. All other marks certainly have a higher error in determination, and if they are to be used for an estimate of the duration of the mitotic phases, this uncertainty has to be kept in mind. The time for each cleavage from the first to the tenth is almost the same in different eggs, provided that a constant temperature is main- tained and the oxygen supply is sufficient. During the process of cleavage the cell divisions follow each other without a typical resting stage, therefore the cleavage time was measured from the breakdown of the nucleus to the breakdown of the daughter nuclei. In Table I the results are compared with those of Jordan and Eycleshymer (1894) on amphibian blastomeres. The numbers concerning the zebra fish egg are all averages of at least 10 eggs. It can be seen from Table I that in every species the divisions show a characteristic duration. In four of the six animals the divisions show a trend towards acceleration before they finally begin to slow down. (The more complicated curve for the Amblystoma egg cannot be discussed here.) The turning point for this trend comes at different times. In the egg of the zebra fish the acceleration is at its height during the fifth cleavage. It seems significant that this is the last division when only one cell layer is involved, for the sixth cleavage is horizontal and divides the blastoderm into two layers. The sixth cleavage takes a slightly longer time than the preceding division, and from then on the process of cleavage grad- ually becomes slower and slower. Acceleration and retardation seem to involve the whole mitotic process uniformly and not any of its 86 EDWARD C. ROOSEN-RUNGE phases differentially. Only during the ninth and tenth cleavages has a prolonged interkinetic phase been recorded, but as the error in deter- mining this phase is even greater than for any other, no conclusion can be drawn from observations made at these stages of development. TABLE I Duration of cleavage divisions in amphibian and teleost eggs.* The times enclosed in brackets refer to individual cases and are not averages. Temperature, C. Ambly- stoma punctatum Rana palustris Diemec- tylus viridescens Bufo variabilis Epiplaly chaperi Brachy- danio rerio 18 18 18 18 24 25 Duration of Cleavage Divisions Fertilization to first cleavage 10 hrs.? 4-5 hrs. 10 hrs. 4-5 hrs. 25 min.? First to second cleavage 1 hour 1 hour 2 hrs. 1 hour 2 hrs. 20 min. Second to third cleavage 50 min. 1 hour 15 min. 1 hour 1 hour 5 min. 1 hour 2 min. 44 min. 19| min. Third to fourth cleavage 55 min. 2 hrs. 15 min. 45 min. 1 hour 1 hour (43 min.) 19 min. Fourth to fifth cleavage 1 hour 40 min. 1 hour 1 hour (41 min.) 18 min. Fifth to sixth cleavage 40 min. (1 hour 50 min. (2 hrs. (39 min.) \1\ min. Sixth to seventh cleavage . ... 35 min.) (1 hour 45 min.) (2 hrs. (39 min.) 18^ min. Seventh to eighth cleavage 25 min.) (1 hour 45 min.) (2 hrs.) (40 min.) 19 min. Eighth to ninth cleavage 25 min.' (1 hour 20 i min. Ninth to tenth cleavage 25 min.) 20 min. * The data on amphibian eggs are taken from Jordan and Eycleshymer (1894). In measuring the relative duration of the mitotic phases every cleavage can, of course, be observed. Most observations, however, were made during the sixth to ninth cleavages, since these stages had to be studied also for the periodicity of divisions, which will be dis- cussed later. The arbitrary definition of the stages is obviously KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 87 a matter of terminology so long as the fundamental mechanism of mitosis is not understood. The prophase was denned as extending from the first swelling of the nucleus until the break-up of the membrane. The time from the break-up until the chromosomes reappeared was assumed to be the duration of the metaphase plus the anaphase. As to the duration of both of these phases, it can only be stated that the metaphase is much shorter than the anaphase. This is true for two reasons, namely: (1) Nuclei which were observed up to the breakdown of the membrane and then immediately fixed always showed the chromosomes already slightly apart, and (2) the very rapid passing of the metaphase as de- TABLE II Duration of mitosis and mitotic phases. Material Total dura- tion Pro- phase Meta- phase Ana- phase Telo- phase Author Protozoon : Rhagostotna schilssleri min- utes 32.5 179 150 180 35 16 18 per cent 18.5 22.5 19.5 20.0 18.5 16.5 per cent 12.5 14.0 12.0 i per cent 18.5 3.5 40.0 19.5 per cent 53.5 60.0 46.5 50.0 i after Darlington after Darlington Jolly Wassermann (after Jolly) Strangeways Roosen-Runge Protozoan : Ruglypha sp Erythrocytes, Triton The same Chorioidea, cartilage in chicken, culture i 80.0 i 26.0 33.5 Blastomeres, Brachydanio . . . The same, interkinetic phase counted as telophase i 55.5 i 50.0 scribed can actually be seen in the egg of Epiplaty chaperi. From these observations it must be concluded that the metaphase probably takes not much longer than one minute, or about 5.5 per cent of the total division time. The telophase was measured from the appearance of the chromo- somes until the nuclei were completely rounded, with only a few partitions within. It was assumed that the reappearance of the chromosomes in life actually indicated a break in the process of mitosis, inasmuch as they become visible at the moment when they begin to take up fluid and pass from more or less solid bodies into vesicles. In Table II, some results on the relative duration of mitotic phases in various animals have been compared. They were all obtained by direct observation. Interesting data like those of Lewis and Lewis EDWARD C. ROOSEN-RUNGE (1917) have been omitted since they are given too inaccurately for the present purpose. They seem, however, not to be in general disagree- ment with the figures presented here. The significance of the data compared lies in the fact that they agree surprisingly well, in spite of the different kinds of material used by the different investigators as well as the great disparity in the observations made with relation to the total duration of mitosis and the definition of its phases. The relative time for the prophase varies only from 18.5 to 22.5 per cent in cells as different as those of protozoa, chicken cartilage, and fish blastomeres. The reported times of the metaphase vary also only slightly. However, in both the anaphase and the telophase there is considerable variation although it is smaller in the anaphase than in the telophase. TABLE III The duration of cleavage divisions under different temperatures.* The times are minutes. Cleavage 2 3 4 5 6 7 8 9 10 23 C. (21) 23| (20) (20) (21) 24 20 19.5 24^ 21 19 (20) (19) 25 19.5 18.5 18.5 18 18.5 19 19.5 20 254 (18) 18 17 17 18 26 (17) 17.5 18 20 26| (16) * The times enclosed in brackets refer to individual cases and are not averages. The telophase in Triton erythrocytes is reported to take 50 per cent of the total time of mitosis, and in protozoa 53.5 and 60 per cent. Lewis and Lewis state that the telophase "which can be more ac- curately recorded than the other phases, shows a striking similarity in all types of cells and much less variation." If we take their telophase and reconstruction periods together as corresponding to the definition of the telophase used here, we find that the telophase in cultures of chicken mesenchyme and smooth muscles lasts about 50 per cent of the whole time of division, while the telophase of the zebra fish blasto- meres takes only about half of this relative time, that is, 26 per cent. Even if the interkinetic phase, the delimitation of which is not at all clear, is added quite arbitrarily to the telophase, there is not more than 33.5 per cent of duration time accounted for. The certainty with which this result is obtained leads to the conclusion that the rela- tive shortness of the telophase is actually significant for the type of karyokinesis we are dealing with, which involves the formation of KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 89 chromosomal vesicles in the telophase, and has no actual "resting phase." The effect of different temperatures on the duration of cleavage is shown in Table III. The results cover only a part of the large range of temperature which the eggs can stand. The only conclusions, therefore, which can be safely drawn are that the duration of cleavage divisions is influenced by even slight changes in temperature, but that the general trend of acceleration for the first six cleavages, and the following retardation, are practically unchanged so that the duration of mitosis may be said to be constant under constant conditions. Many investigators, however, have found the duration of mitosis varying up to several hundred per cent for the same kind of cells. Observations of cells in tissue cultures in particular have yielded results which were very inconsistent with respect to the total duration of division. In all these cases the inconsistency can be attributed only to varying conditions of nutrition, oxygen supply, and temperature. A comparison of the results given in the literature and the observations on the eggs of the zebra fish, show that under constant conditions the duration of mitosis is constant and characteristic for the different types of cells. DISCUSSION The process of cleavage is characterized by continuous and often synchronous cell divisions, which frequently follow a definite pattern. In general there is no morphological differentiation during cleavage, but very often there is a segregation of different materials in different cells. At the end of the cleavage period there is a break in the develop- ment, the divisions cease to be continuous and synchronous, and the period of cell migration and arrangement begins, often together with the first histological differentiation. On the other hand, cleavage is regarded as "but a continuation ... of that series of cell-divisions which has been going on uninterruptedly, though with periodic pauses, since the most remote antiquity. The divisions of the egg during cleavage are in all essentials of the same type as those of adult cells; such differences as may appear e.g., the prominence of asters, the frequent asymmetry of the amphiaster, and the consequent inequality of cleavage are of minor importance, though often interesting for analyzing the mechanism of mitosis." (E. B. Wilson, 1928, page 981.) The general conception is that cleavage divisions are dynamically somewhat different from the divisions in the older animal, but that their variation is not correlated with any essentially different mecha- nism. There are, however, observations which point to a difference in mechanism. Investigators of the chromosomal vesicles, which so 90 HOWARD C. ROOSEN-RUNGE frequently occur in the telophase of cleavage divisions, have often suspected that this particular feature of mitosis might be immediately connected with the fact that cleavage divisions go on continuously and almost without interphases. The study of karyokinesis in zebra fish blastomeres reveals that the formation of chromosomal vesicles is obviously in itself a process of much shorter duration than the common type of telophase and, further- more, that it represents a condition which permits of an almost im- mediate start of the next division without a "resting stage" and with- out a spireme formation in the prophase. No nucleoli are formed in this type of mitosis. All these features are characteristic only for the divisions during cleavage. About the time of the twelfth cleavage an entirely different type of mitosis appears, which shows no chromosomal vesicles in the telophase, but nucleoli and a very distinct spireme in the prophase (Fig. 5). In my material no transitional forms have been observed between these two types, though it is quite possible that a more thorough investigation may reveal such transitions. Chromosomal vesicles have been found in the eggs of very many species and almost all classes of animals with the possible exception of birds and mammals. (A review of the literature has been given by Richards, 1917.) The suggestion seems obvious that the type of mitosis which is characterized most strikingly by the formation of chromosomal vesicles in the telophase, is due to some aspect of the division mechanism that is peculiar to the cleavage divisions. We have not yet arrived, however, at any definite conclusions concerning the possibly different dynamics involved. SUMMARY The nuclei in the blastomeres of Brachydanio rerio can be observed easily in life. They are visible in the prophase and telophase as well as in the interkinetic phase. This discovery is used (1) to confirm and consolidate the results obtained from sectioned material; (2) to fix the blastomeres in any desired mitotic phase; and (3) to determine the duration of mitosis and its phases. The duration of mitosis and its phases under constant conditions, particularly with respect to temperature, is found to be constant for each cleavage. The time from the breakdown of 32 nuclei to the break- down of 64 nuclei is 18 minutes at 25 C. This places the cleavage divisions of the zebra fish among the most rapid ever observed. The first six cleavages show a trend towards acceleration, the sixth being the most rapid one. From then on the speed of the divisions slows down. This trend is essentially undisturbed by changes in temperature. KARYOKINESIS DURING CLEAVAGE OF ZEBRA FISH EGG 91 The nuclear divisions during cleavage are characterized (1) by the formation of chromosomal vesicles in the telophase (some of these vesicles can frequently be seen in life to persist through the interphase) ; (2) by a comparatively short duration of this type of telophase; (3) by a very short, if any, true interphase; (4) by the lack of nucleoli; and (5) by the absence of a typical spireme formation in the prophase. The very short duration of the telophase has been recorded for the first time. The other observations have been found in the cleavage divisions of a majority of the species examined. In the zebra fish egg they continue until about the twelfth cleavage, when the form of mitosis typical for the adult first appears. It is suggested that this type of mitosis is probably associated with the rapid sequence of divisions and is generally characteristic of cleavage mitoses. The most characteristic feature of this type of karyokinesis is the formation of the chromosomal vesicles, but the shortening of the interphase and telophase, and the lack of spireme formation in the prophase are also obvious. I am indebted to Professor J. W. Wilson, Brown University, for his very valuable advice and to Professor H. E. Walter for his assistance in editing the paper. LITERATURE CITED DARLINGTON, C. D., 1937. Recent Advances in Cytology. Philadelphia. JOLLY, J., 1904. Recherches experimentales sur la division indirecte des globules rouges. Arch. Anat. Micros., 6: 455. JORDAN, E. O., AND A. C. EYCLESHYMER, 1894. On the cleavage of amphibian ova. Jour. Morph., 9: 407. LEWIS, W. H., AND M. R. LEWIS, 1917. The duration of the various phases of mito- sis in the mesenchyme cells of tissue cultures. Anat. Rec., 13: 359. MOENKHAUS, W. J., 1904. The development of the hybrids between Fundulus heteroclitus and Menidia notata with especial reference to the behavior of the maternal and paternal chromatin. Am. Joiir. Anat., 3: 29. RICHARDS, A., 1917. The history of the chromosomal vesicles in Fundulus and the theory of genetic continuity of chromosomes. Biol. Bull., 32: 249. RICHARDS, A., 1935. Analysis of early development of fish embryos by means of the mitotic index. I. The use of the mitotic index. Am. Jour. Anat., 56: 355. RoosEN-RuNGE, E. C., 1938. On the early development bipolar differentiation and cleavage of the zebra fish, Brachydanio rerio. Biol. Bull., 75: 119. SMITH, B. G., 1929. The history of the chromosomal vesicles in the segmenting egg of Cryptobranchus allegheniensis. Jour. Morph., 47: 89. STRANGEWAYS, T. S. P., 1922. Observations on the changes seen in living cells during growth and division. Proc. Roy. Soc. London, Series B., 94: 137. WASSERMANN, F., 1929. Wachstum und Vermehrung der lebendigen Masse. Handb. der mikrosk. Anat., 1: 2. WILSON, E. B., 1928. The Cell in Development and Heredity. MacMillan Co., New York. THE EFFECTS OF LIGHT AND TEMPERATURE ON THE MALE SEXUAL CYCLE IN FUNDULUS SAMUEL A. MATTHEWS (From the Thompson Biological Laboratory, Williams College} Fundulus heteroditus is a teleost fish which breeds during the late spring and early summer months. Its gonads undergo fairly definite seasonal changes, reaching their greatest weight just before the breed- ing season, falling off sharply just after spawning is completed in late July, then undergoing a period of slow growth until the onset of rapid maturation of germ cells prior to the next breeding season (Matthews, 1938). Several factors, acting independently or collectively, may be concerned in the control of this gonad cycle. Of these factors the pituitary gland as an internal factor and temperature and light among the external factors might reasonably be supposed to possess some degree of control. Evidence concerning the role that the pituitary gland plays in the sexual cycle has already been obtained (Matthews, 1939). The following experiments are concerned with the effects of light and temperature on the male sexual cycle. From the experiments of a number of workers, particularly those of Bissonnette (see his review article, 1936) it is clear that in some birds and mammals light plays a dominant role and temperature a subordinate one in controlling the seasonal cycle in the gonads. The data on poikilothermous animals are not as conclusive. Clausen and Poris (1937) in the case of Anolis and Burger (1937) in the case of Pseudemys both believe that light is important in controlling the sexual cycle. Bellerby (1938), on the other hand, finds no evidence "that light is essential for the maintenance of reproductive activity in Xenopus laevis or that seasonal variation in light intensity or wave length plays any part in the control of the sexual cycle under natural conditions." Turner (1919), in describing seasonal changes in the spermary of the perch, pointed out that tremendous synthesis of material in the testis occurs in August when the temperature of the water has reached its peak and begun to decline, and expulsion of the sperm occurs when the temperature begins to rise. No experiments controlling the light factor were described. From his work with the stickleback Craig-Bennitt (1930) also concludes that temperature is the important factor in controlling the sexual cycle and that light is 92 LIGHT AND TEMPERATURE EFFECTS ON FUNDULUS 93 unimportant. More recently Hoover and Hubbard (1937) have shown that a gradual increase in daily illumination followed by a gradual decrease will cause brook trout (a fall breeding animal) to produce ripe eggs and mature sperm several months earlier than normal. To determine whether or not the absence of light exerts any in- hibitory influence on spermatogenesis in Fundulus the following two experiments were carried out. In the first, begun in December, 11 males were divided in such a way that 5 were maintained in an aquarium subjected to ordinary daylight with no night illumination and the other 6 were kept in a light-proof tank. They were fed daily with the stock food used in all experiments, consisting of dried shrimp, puppy biscuit and Mead's infant cereal, with occasional living food such as worms or Daphnia. These animals were killed at five different in- tervals over a period of 3 weeks. The average percentage of the body weight formed by the testis of the animals in the light-proof tank was 0.56 per cent, that in the other aquarium 0.37 per cent, and microscopic examination of the testes showed no significant differences in the state of activity of the two groups. In the second experiment, begun in March and extending into April, 4 males were placed in the lighted aquarium, which in this instance was illuminated at night by a 50-watt mazda bulb suspended above the tank, and 8 were kept in the dark. The animals were killed over a period of 4 weeks. The percentage of the body weight formed by the testis in the illuminated aquarium averaged 1.76 per cent, in the darkened tank 1.88 per cent, and again no structural differences were observed in the microscopic structure of the two groups of testes. In these cases some of the animals killed during April presented the white testis and numerous sperm asso- ciated with a high degree of activity, and this occurred as early in the darkened tank as in the illuminated one. From these experiments it seems fairly clear that absence of light for at least 4 weeks prior to the breeding season does not inhibit activation of the testis in Fundulus. It should, of course, be noted that the animals which developed ripe testes in the dark had been on a rising daylight curve for nearly three months before they were placed in the dark. Whether or not this is a significant factor in initiating the active phase in the testis cycle has not been determined. Experiments concerning the effect of temperature on activation of the testis gave somewhat different results. Ten animals were kept in a tank in which the temperature of the water averaged 21 C. (variation 19-21.5) and 10 animals were kept in a constant temperature room with light conditions similar to those of the first group, the temperature of the water in the aquarium here averaging 5.5 C. (variation 4-7 C.). 94 SAMUEL A. MATTHEWS In a series run during December the testis formed as large a percentage of the body weight of animals in the cold room as in the normals (0.53 per cent), but sections of the testes of animals after 23 days in the cold room showed that these were retarded in development, particularly in the later stages of spermatogenesis. In a series run during March and April, moreover, the testes of those maintained at 5.5 C. averaged only 1.16 per cent of the body weight as against 1.76 per cent for those in the warmer room and in general animals maintained in the cold produced sperm much later than did those in the warm room. The retarding effect of the low temperature was noted in this series as early as 9 days after the beginning of the experiment. Only one individual, killed April 5 after 21 days in the lower temperature, showed a degree of activity comparable with that of the control animals. In brief, then, records have been obtained on 14 animals main- tained in a light-proof tank from 4 to 55 days as compared with con- trols subjected to daylight or to daylight and added night illumination. The testes of these animals were like those of the controls and in cases killed late in April those of both groups were whitish and filled with sperm. Only one case, killed December 26 after 23 days in the dark, showed a testis less developed than that of the control. Records have also been obtained on 17 animals which were maintained at a tempera- ture of 5.5 C. as compared with 13 control animals kept at 21 C. After 9 to 23 days at the lower temperature spermatogenesis was definitely retarded. These experiments show that the presence of light is not essential for complete activation of the testis of Fundulus; and that low tem- peratures exert a retarding influence on maturation of the sperm. Obviously no evidence is furnished concerning the effects of gradual changes in the amount of light to which the animal is subjected daily, which Hoover and Hubbard found of such importance in the sexual cycle of the trout. LITERATURE CITED BELLERBY, C. W., 1938. Experimental studies on the sexual cycle of the South African clawed toad (Xenopus laevis). II. Jour. Exper. Biol., 15: 82-90. BISSONNETTE, T. H., 1936. Sexual photoperiodicity. Quart. Rev. Biol., 11 : 371-386. BURGER, J. W., 1937. Experimental sexual photoperiodicity in the male turtle, Pseudemys elegans (Wied). Am. Nat., 71: 481-487. CLAUSEN, H. J., AND E. G. PORIS, 1937. The effect of light upon sexual activity in the lizard, Anolis carolinensis, with especial reference to the pineal body. Anat. Rec., 69: 39-54. CRAIG-BENNITT, A., 1930. The reproductive cycle of the three-spined stickleback, Gasterosteus aculeatus, Linn. Phil. Trans. Roy. Soc. London, Series B., 219: 197-279. LIGHT AND TEMPERATURE EFFECTS ON FUNDULUS 95 HOOVER, E., AND H. E. HUBBARD, 1937. Modification of the sexual cycle in trout by control of light. Copeia, 1937: 206-210. MATTHEWS, S. A., 1938. The seasonal cycle in the gonads of Fundulus. Biol. Bull., 75: 66-74. MATTHEWS, S. A., 1939. The relationship between the pituitary gland and the gonads in Fundulus. Biol. Bull., 76: 241-250. TURNER, C. L., 1919. The seasonal cycle in the spermary of the perch. Jour. Morph., 32: 681-711. SOME EXPERIMENTS ON THE RELATION OF THE EXTERNAL ENVIRONMENT TO THE SPERMATO- GENETIC CYCLE OF FUNDULUS HETEROCLITUS (L.) l J. WENDELL BURGER (From Trinity College, Hartford, Connecticut, and The Ml. Desert Island Marine Biological Laboratory, Salsbury Cove, Maine) INTRODUCTION Within the last decade a considerable body of experimental work has shown that the sexual cycles of many vertebrates of the north temperate zone are regulated in part by the annual cycle of changes in day-length. Little, however, is known about the relation of the ex- ternal environment to the sexual cycle of cold water fish. Success in modifying the piscine sexual cycle by light agencies has been reported for the trout (Hoover, 1937; Hoover and Hubbard, 1937), for the minnow, Phoxinus (Spaul cited from Rowan, 1938), and for the stickle- back (Tinbergen cited from Rowan, 1938). Craig-Bennett (1930) came to the conclusion that the sexual cycle of the stickleback was regulated primarily by temperature. HoQver (private communication to T. H. Bissonnette) has found that light is ineffective on yellow perch which were kept in water below 44 F. The normal sexual cycle of Fundulus has been described by Mat- thews (1938). As in many cold-blooded vertebrates the sexual cycle is a continuous process throughout, with no genuinely inactive phase, although during the winter there is little or no spermatogenetic activity. In the late summer and fall a limited production of spermatogonia takes place. Vigorous spermiogenesis begins in the spring, with a mating period during May and June. Thereafter occurs a gradual deceleration of spermiogenesis with a concomitant testicular involution. It is noticed that the major portion of the spermatogenetic activity is present during the spring when the days are increasing in length, and when the temperature of the water is rising. The experiments here reported are to test the relation of light and temperature to the spermatogenetic cycle of Fundulus. 1 Aided in part by a grant from the American Philosophical Society administered by T. H. Bissonnette for 1938-39. 96 SPERMATOGENESIS IN FUNDULUS 97 MATERIALS AND METHODS Over seven hundred newly captured adult male Fundulus were used in four experiments. Two of these experiments were performed in Maine, and two in Connecticut. Fish were secured on June 30 from a tidal inlet on Mt. Desert Island, Maine, and were confined to laboratory aquaria fed by sea water. One control aquarium was placed out-of- doors in a well lighted spot. The fish therein were maintained on natural daylight until August 27. Two other aquaria were placed in a light-proof box, which was illuminated by two 50-watt lamps. From June 30 to July 22 the daily light ration was reduced 20 minutes per day from 15 hours to 8 hours. Then between July 22 and August 27 the daily light ration was increased 20 minutes per day from 8 hours to 20| hours. The temperature of the aquaria water ranged between 11 and 17 C., in general increasing in warmth from June to August. On October 29 Fundulus were secured from a tidal inlet off Long Island Sound near Niantic, Connecticut. These animals were placed in fresh water aquaria at Hartford, Connecticut. The control fish were exposed to daylight for natural day-lengths between October 29 and January 4. The experimental fish received in addition to natural daylight illumination from a 100-watt lamp. The exposure to electric light was increased every 5 days so that by December 1 1 the fish were receiving 8| hours of electric illumination added to normal daylight. After this date no further increases in the length of time of exposure to light were made. The temperature of the fresh water aquaria ranged between 11 and 18 C., decreasing in warmth from October to January. In a final experiment fish were captured from a tidal inlet at Old Lyme, Connecticut, on February 25, and were confined to fresh water aquaria at Hartford. All aquaria were made light-proof. The fish never received more than 1| hours of light per day during the experi- mental period which was from February 25 to March 25. This limited exposure to light was necessary for feeding. One group of males were kept in cold water which varied in temperature between 6 and 10 C. Another group was kept in warmer water which ranged between 14 and 20 C. There was always at least 6 C. difference in temperature between the two groups. Fish were also sampled from the wild at various intervals. The animals were fed almost daily on either chopped livers or clams. Only healthy, fungus-free fish were killed for histological study. 98 J. WENDELL BURGER RESULTS Confinement to aquaria and to fresh water had no deleterious effects on the testes. Judging from the condition of the internal organs, the diet was more than adequate for the maintenance of good health. In all the experiments with light, and where there was no significant difference in temperature between controls and experimentals, no dif- ference in the state of the testes was found between control and experi- mental fish. The experimental light rations were: 21 days of gradually decreased lighting between June 30 and July 22; 37 days of gradually increased lighting between July 22 and August 27; and 68 days of gradually increased lighting between October 29 and January 4. On June 30 at the start of the first experiment, spermiogenesis was at its peak. As can be seen in Fig. 1, there exists at this time a broad zone of cortically located spermatogonia etc. surrounding medullary tubules which are filled with sperm. During the summer there occurs a gradual decrease in the proliferation of spermatogonia, together with a loss of sperm in the tubules. The rate of this testicular involution can be seen by comparing Figs. 1,2, and 4. Figures 2 and 4 are from laboratory control fish for July 22 and August 27 respectively. That the fish which received 21 days of gradually decreased lighting showed no differences in testicular state when compared with control fish, is illustrated in Figs. 2 and 3. Figure 2 is from a control testis, and Fig. 3 from an experimental fish whose light ration was gradually reduced to 8 hours per day. Likewise a comparison of Figs. 4 and 5 shows that there was no difference in testicular state between control fish (Fig. 4) and experimental fish (Fig. 5) after 37 days of gradually increased lighting applied to Fundulus which previously experienced 21 days of decreased lighting. These results indicate that the testicular involution which normally occurs during the summer when the day-lengths are naturally shortened cannot be hastened by 21 days of decreased lighting. Moreover, 37 days of subsequent increased lighting does not change the rate of testicular involution, nor does it induce a precocious new spermato- genetic cycle. Since many animals are refractory to photoperiodic manipulations at the end of their sexual cycle, the above experiments are no test for the efficacy of photoperiodic manipulations on the sexual cycle. The fish which were lighted for 68 days between October 29 and January 4 offer a fair test, however, as to whether or not the sexual cycle of Fundulus can be influenced by light. When this experiment was begun, the cortical zone of the testis was slowly proliferating spermato- gonia, while the medullary system of tubules was involuted and devoid PLATE I C-, ** '- F^ ;;''. rt-, 5 xv;'m^ ? All figures are unretouched photomicrographs, X 80. FIG. 1. Section of a testis from a fish captured 6/30/38. The cortical zone (at the top of the figure) of spermatogonia, etc. is broad; the medullary zone of tubules is black with sperm. FIG. 2. Section of a testis from a laboratory control, 7/22/38. The spermato- gonia are fewer than in Fig. 1, and the tubules contain fewer sperms. FIG. 3. Section of a testis, 7/22/38 after 21 days of shortened day-lengths. The testis is in the same condition as that of the controls (Fig. 2). FIG. 4. Section of a testis from a laboratory control, 8/27/38. Spermiogencsis is almost finished; the tubules have markedly involuted. FIG. 5. Section of a testis, 8/27/38 after 37 days of increased lighting. No significant difference is found between this and the control (Fig. 4). 100 J. WENDELL BURGER of sperm. This condition can be seen in Fig. 6, which is a section of a testis on October 29. During this experiment sperm were produced both by the control and experimental fish. These sperm can be seen in Fig. 7 which is from a control fish on January 4, and in Fig. 8 which is from an experi- mentally lighted fish on January 4. In the control fish, sperm were formed while the days were decreasing in length, as indicated by fish sampled in December. In the experimental fish, sperm were formed no more abundantly when the day-lengths were increased in length by means of 8| hours of electric light added at night. Thus for fish at the threshold of a new spermatogenetic cycle, the application of increased or decreased daily rations of light does not modify the rate of the subsequent formation of sperm. A comparison of laboratory fish and fish from nature in early January showed that the fish in their natural habitat do not form sperm at this time as did the laboratory fish. This statement needs to be qualified slightly for there are Fundulus in nature which during the winter form a very few spermatozoa. However, the general condition for winter fish at least up until early March is similar to that shown in Fig. 6. The most obvious difference between the laboratory fish and the fish from nature is the difference in water tem- peratures. The laboratory fish lived in water between 11 and 18 C., while the fish in nature during the winter lived in water whose tem- perature was near C. The experiment with temperature where the daily light ration was only long enough for feeding the fish indicates that spermatogenesis is responsive to temperature manipulations. Figure 9 is a section from a testis from a fish after 29 days in water whose temperature varied between 6 and 10 C. These temperatures were somewhat higher than those experienced by the fish in nature at the time of capture and during the experimental period. This testis is a winter testis and shows no transformations of spermatozoa. However, there did occur a slight multiplication of spermatogonia so that the testis was not completely inactive during this period. Figure 10 is a section of a testis from a fish after the same 29 days in water whose temperature varied between 14 and 20 C. Here spermatozoa have been formed in large numbers. This effect of higher temperature can readily be seen by comparing Figs. 9 and 10. This result was uniform for all fish. It should be emphasized that these fish never had more than 1| hours of light per day during the experimental period, and usually not more than one- half hour. PLATE II !l ' IMYftS ^4pi75?--v"~ -,~ . fc^ r-~ .-*'. _ jc-^ , FIG. 6. Section of a testis from a fish captured 10/29/38. The bulk of the testis consists of spermatogonia. Sperms are absent and the tubular system is greatly reduced. FIG. 7. A section from a laboratory control, 1/4/39. The black areas are tubules which contain sperm. FIG. 8. A section of a testis, 1/4/39 after 68 days of increased lighting. Sperms while abundant are no more numerous than in the controls (Fig. 7). FIG. 9. A section of a testis, 3/25/39 after 29 days in almost complete darkness in water whose temperature varied between 6 and 10 C. This is essentially a winter testis consisting of spermatogonia (compare with Fig. 6). Sperms are absent. FIG. 10. A section of a testis, 3/25/39 after 29 days in almost complete darkness in water whose temperature varied between 14 and 20 C. The black areas show the large numbers of sperms that have formed. 102 J. WENDELL BURGER DISCUSSION These experiments indicate that light as such is of no importance in the spermatogenesis of Fundulus. Spermatozoa can be formed in almost complete darkness, and will form in equal abundance when the days are either increasing or decreasing in length. Temperature appears as the important factor of the external environment which modifies spermatogenesis. In cold water spermatogenesis is retarded or inhibited, while in warm water spermatogenesis is rapidly completed. These experiments give no exact data on the critical temperatures involved, but from our observations both on experimental fish and on fish in nature a general scheme seems clear. At temperatures near C. the testis is inactivated. As the temperature rises toward or around 10 C. spermatogonial multiplications occur. Still higher tem- peratures permit the transformations of sperm to take place. Marine temperatures show a very orderly annual cycle. Dr. R. A. Goffin kindly gave us the mean daily sea water temperatures for 1938 at Woods Hole, Massachusetts. Dr. V. L. Loosanoff also referred us to his paper (Loosanoff, 1937) which gives the shallow water tempera- tures for over three years at Charles Island, Long Island Sound. Both sets of data show a low point in February followed by a continued rise beginning in March and reaching a maximum in August. From August to September a continued drop takes place. The spermatogenetic cycle of Fundulus fits nicely into this annual temperature curve. In the fall as the temperature drops spermato- gonial multiplications take place. During the winter the testis is relatively inactive. In fact, fish captured in late February show slightly less testicular activity than those captured in early January. The spring rise in water temperature is accompanied by increased spermatogenetic activity. It should be remembered that in nature the beginning of active spermatogenesis coincides with the warming of the water, and not with the increased lengthening of the days which began three months previously. SUMMARY AND CONCLUSIONS 1. No differences in the velocity of the spermatogenetic cycle of adult male Fundulus were found between control and experimental fish kept in water of the same temperature when treated as follows: (a) 21 days of gradually decreased day-lengths between June 30 and July 22, (b) 37 days of gradually increased lighting subsequent to treatment as in (a) between July 22 and August 27, (c) 68 days of in- creased lighting between October 29 and January 4. 2. Laboratory fish kept during the late fall and early winter in SPERMATOGENESIS IN FUNDULUS 103 water whose temperature was higher than that experienced by fish in nature showed an acceleration of spermatogenesis. 3. Laboratory fish which received no more than 1| hours of light per day and which were kept in water of from 6 to 10 C. between February 25 and March 25 remained inactive sexually. Fish which received no more than 1^ hours of light per day and which were kept in water whose temperature varied between 14 and 20 C. formed large numbers of sperm within this same period of time. 4. It is concluded: (a) that the spermatogenetic stages of the annual sexual cycle are not affected by light as light; (b) that the temperature of the water is the important factor of the external environ- ment regulating spermatogenesis in Fundulus. 5. It is suggested that at temperatures near C. sexual activity is inhibited. As the temperature rises toward or near 10 C. sperma- togonial multiplications occur. Still higher temperatures produce complete spermatogenesis. LITERATURE CITED CRAIG-BENNETT, A., 1930. The reproductive cycle of the three-spined stickleback, Gasterosteus aculeatus, Linn. Phil. Trans. Soc., Series B, 219: 197-279. HOOVER, E. E., 1937. Experimental modification of the sexual cycle in trout by control of light. Science, 86: 425-426. HOOVER, E. E., AND H. E. HUBBARD, 1937. Modification of the sexual cycle in trout by control of light. Copeia, 1937: 206-210. LOOSANOFF, V. L., 1937. Spawning of Venus mercenarius (L). Ecology, 18:506-515. MATTHEWS, S. A., 1938. The seasonal cycle in the gonads of Fundulus. Biol. Bull., 75: 66-74. ROWAN, WM., 1938. Light and seasonal reproduction in animals. Biol. Rev., 13: 374-402. INFLUENCE OF THE SINUSGLAND OF CRUSTACEANS ON NORMAL VIABILITY AND ECDYSIS 1 F. A. BROWN, JR., AND ONA CUNNINGHAM (From the Zoological Laboratory, Northwestern University) Since the work of Perkins and of Roller in 1928, who independently described the presence of a substance in eyestalk extract which exer- cises a very potent effect upon the chromatophores of crustaceans, there has been much interest shown in the crustacean eyestalk func- tion. The picture has been rendered even more interesting as a result of the work of Brown (1935) and of Kleinholz (1938), demonstrating that humoral activity in this group of animals is by no means a simple one, but that several hormonal substances are normally functioning. Hanstrom (1935) performed experiments in which he showed that the portion of the eyestalk which was active in affecting chromatophores always contained, among other things, a tissue which he has termed the sinusgland. This has given rather good evidence indicating which tissue of the eyestalk is the active one in this regard. The cells of this tissue were shown to be secretory in nature and to contain a rich supply of secretory granules. The more recent work of Hanstrom (1936), Stahl (1938) and others have shown the sinusgland to be present in some degree in all the crustaceans that have been examined in detail. Its occurrence appears to be quite independent of the state of development of a chromatophore system. Functionally it appears to have common properties with the corpora allata of insects since an extract of the latter organ in many cases serves as an activator of crustacean chromatophores. Abramowitz (1936, 1938) has demon- strated that the chromatophorotropic substance from the sinusgland and the intermedin of the vertebrates have certain common chemical and physiological properties. Koller (1930) was the first to demonstrate that the eyestalk sub- stance has another function in addition to the control of chromato- phores. He found that animals from which the eyestalks had been removed failed to deposit calcium in their exoskeletons to the same extent as normal animals. He interpreted this to be the result of removal of the source of a controlling hormone. Welsh (1937) found that when he perfused an exposed crayfish heart with eyestalk extract 1 This investigation was supported by a research grant from the Graduate School of Northwestern University. 104 CRUSTACEAN SINUSGLAND AND VIABILITY 105 there was a pronounced speeding up of that organ. Brown (1938) demonstrated that removal of the eyestalks appreciably shortened the life of the individual and that the shortening thus induced could be compensated for in part by implantation of eyestalk tissue into the ventral abdomen. This shortening of the life of the animal has been called a "viability effect " of an eyestalk hormone, though it is fully realized that this is a function described in far too general terms. It is hoped that this "viability effect" can soon be analyzed into the particular phenomena responsible for the shorter life. There has frequently been suggestion of a "molting effect" of the eyestalk substances, though no adequate data have yet been published to establish such a function. The only grounds for such a belief are that several investigators have mentioned that eyestalkless animals appear to molt more frequently than normal ones. No reason has been advanced for thinking the effect is due to anything other than the injury caused by the operation of eyestalk removal (indicated by Darby, 1938). The following research has been conducted in continuation of that of Brown (1938) with the intention of discovering just what tissue of the eyestalk is responsible for the "viability effect" of this organ. There is included here the first direct evidence for an endocrine activity of the sinusgland of the crustacean. Hitherto its functioning had been supposed upon the grounds of the best of circumstantial evidence. During these experiments the sinusgland has been dissected out and implanted into the ventral abdominal sinus of eyestalkless animals. Direct physiological evidence of its endocrine function has been dem- onstrated. Furthermore, it is quite well established as a result of these experiments that this gland is the one responsible for the normal continuation of life of the animal and also that it has a functional activity in the control of molting. The possibility of explaining the viability effect of eyestalk hormones in terms of molt control will be discussed. METHODS AND MATERIALS All the crayfishes used in these experiments were small individuals (carapace lengths 15-30 mm.) of the species Cambarus immunis, with the exception of certain large individuals (Cambarus virilis, C. blan- dingii, and C. immunis of carapace lengths 30-40 mm.) which were used as the source of the sinusgland for implantation. The animals were brought into the laboratory a few days before the beginning of an experiment. It was our purpose to use experimental extirpation and implantation to determine the normal functions of the eyestalk gland within the body. 106 F. A. BROWN, JR., AND ONA CUNNINGHAM The method of extirpation was simple : the eyestalks were removed as a whole and the wound sealed with an electric cautery. By so sealing the wound, less than 10 per cent of the animals died as a result of the operation. It is fully realized that such a method of gland extirpation removed much tissue in addition to that of the sinusgland. In the first experiment to be described the implantation consisted of all the eyestalk tissue. The eyestalks were removed from an animal and dropped into amphibian Ringer's solution. With the aid of a dissecting microscope the exoskeleton of the eye was cut away. The soft parts of the eyestalk were easily removed with fine forceps. This tissue was then teased into minute fragments and injected by means of a glass capillary pipette into the ventral sinus of the abdomen. The glass pipette proved to be especially satisfactory since it was possible to ascertain that all of the tissue entered the animal and none was left adhering to the walls of the pipette. In those experiments in which the sinusgland by itself was to be implanted the gland was carefully dissected out in the following man- ner: the eyestalk was removed from a large crayfish and dropped into a watchglass containing amphibian Ringer's solution or a balanced salt solution based on Griffeths' analysis of Astacus blood (which will henceforth be referred to as Griffeths' solution). With a pair of sharp pointed scissors the chitinous exoskeleton was clipped to free the dorsal half of the stalk skeleton from the ventral half. The contents of the stalk were then picked out with fine pointed watch-maker's forceps and the dorsal tissue was teased away in the direct light of a strong lamp. The sinusgland tissue stood out quite conspicuously as a seem- ingly fibrous and granular bluish tissue. This mass of tissue was easily torn away from the adjacent nerve tissue. All the adhering tissue was teased away and the gland rinsed in amphibian Ringer's or Griffeths' solution. With forceps the gland was next pushed through an opening made in the ventral side of the abdomen. The clear exoskeleton in this region made it possible to ascertain that the minute gland was actually left in place upon removal of the forceps. In order to determine the exact location of the tissue removed from the eyestalk, sections were made of the bluish gland-like tissue that was removed, and also of all the remaining portions of the eye- stalk. In addition, longitudinal sagittal sections of the complete eyestalk were made as a control. By study of these three sets of sections it was readily determined just what tissue was being implanted. It was discovered that the implant tissue in histological section ap- peared to be definitely glandular in nature and occupied a position wedged between the medulla externa and the medulla interna. Con- CRUSTACEAN SINUSGLAND AND VIABILITY 107 sidering its position and the fact that its cytoplasm was richly charged with eosinophilic inclusions, it seemed highly probable that this gland was the same as that described by Hanstrom (1936) as the sinusgland. The accompanying photographs show this gland as it occurs in Cam- barus virilis. The first photograph is a median sagittal section of the ^rm t %* *% * FIG. 1. Sagittal sections through the cyestalk of Cambanis virilis (6 micra thick and stained with Delafield's haematoxylin and eosin). A. At a magnification of 80 X, showing the sinusgland as a somewhat triangular section of tissue located dorsally to a point intermediate between the medulla externa and the medulla interna. B. A higher magnification (360 X) of the central region of the sinusgland. 108 F. A. BROWN, JR., AND ONA CUNNINGHAM eyestalk at a magnification of approximately 80 X and the second is a higher power magnification (about 360 X) in the central region of the gland. During the experimental period all the animals were kept in indi- vidual glass finger bowls in water not quite deep enough to cover the carapace. These finger bowls were covered loosely with glass plates to minimize evaporation of the water but still to permit circulation of air over the water surface. The experiments performed included extirpation and implanta- tion, with appropriate controls, and observations were made upon viability and molt behavior. 100 10 15 25 30 35 40 45 TIME IN DAYS FIG. 2. The relation between the percentage of animals dead and the number of post-operative days for eyestalkless crayfishes, (O); eyestalkless crayfishes with a heteroplastic implant of sinusglandless eyestalk tissue, ( o) ; and eyestalkless crayfishes with only a heteroplastically implanted sinusgland, (3 ). EXPERIMENTS ON VIABILITY EFFECTS Experiment I The animals of this experiment, all Cambarus immunis with both eyestalks removed and the stubs cauterized, were divided into three lots. In the first lot were 7 animals with no further treatment. The second lot of 17 animals had a sinusgland taken from a single eyestalk of a large Cambarus virilis or Cambarus bland ingii acutus implanted into the ventral sinus of their abdomens. The third lot of 18 animals had an abdominal implantation consisting of all the eyestalk tissue of a single eyestalk of Cambarus virilis or Cambarus blandingii acutus, from which the gland had been carefully removed. The results of this experiment are best shown in the form of a graph (Fig. 2) in which the percentage of animals dead is plotted CRUSTACEAN SINUSGLAND AND VIABILITY 109 against the post-operational day. This graph demonstrates clearly that eyestalkless animals without abdominal implants live significantly shorter lengths of time than eyestalkless animals into which eyestalk tissue minus the sinusgland has been implanted. Similarly, eyestalk- less animals which have received abdominal implants of the minute sinusgland by itself, live very significantly longer than those animals into which the remaining portion of the eyestalk tissue was implanted. Comparing only the instance of sinusgland implant with the case of no implant, we can conclude definitely that the minute sinusgland lengthens the post-operative life of the animal considerably. It is well to bear in mind that these two latter groups have been subjected to operations of different degrees of severity, in which the animals which 100 10 15 20 25 30 35 40 45 FIG. 3. The relation between the percentage of animals dead and the number of post-operative days for eyestalkless crayfishes, (O); eyestalkless crayfishes with a homoplastic implant of sinusglandless eyestalk tissue, ( )); and eyestalkless cray- fishes with only a homoplastically implanted sinusgland, (3 ). live longer have been subjected to more severe operative injury, the animals of the latter group having their abdomens punctured as well as having both eyes removed. A logical explanation of the inter- mediate length of post-operative life in the instance of those animals with the glandless stalk tissue implants is that there is present in the blood spaces of the general eyestalk tissue a product that has arisen from the sinusgland. During its removal, a bluish liquid is seen to diffuse out of the gland and infiltrate into the surrounding tissues. The general stalk tissue is frequently filled with a homogeneous blue liquid which, in all probability, comes from the same origin. We believe, therefore, that this additional substance is responsible for permitting these animals to live longer than those in which no implant 110 F. A. BROWN, JR., AND ONA CUNNINGHAM is made. It is also possible that fragments of the gland itself still remain which were not removed at the time of operation. The implants in this experiment are heteroplastic, while in an experiment to be described later all the implantations were autoplastic as was the case with those observations published by Brown (1938). It becomes doubly interesting that the sinusgland has a definite effect not only upon the length of post-operative life in the same species of animal, but that the tissue from one species is capable of working effectively within the body of another species to the same end. Thus these substances,- or this substance, is inter-specifically active. Experiment II In this experiment, like the preceding one, eyestalkless Cambarus immunis were divided into three lots. In the first lot, consisting of 6 large animals, there was no further treatment. A single sinusgland from an eyestalk of a large animal of the same species was abdominally implanted into each of the 20 small animals of the second lot. Each of the 20 animals of the third group received an abdominal implant consisting of the tissue from a single large eyestalk from which the gland had been removed. The results of this experiment are shown in Fig. 2. This experiment confirms the influence of the sinusgland on via- bility demonstrated in Experiment I. Here the implantations were homoplastic, from large Cambarus immunis to small Cambarus im- munis. As in Experiment I, the animals without any implant lived a much shorter time than those with sinusgland implants, and animals in which sinusglandless eyestalk tissue was implanted lived for an intermediate length of time. EXPERIMENTS ON THE MOLTING CONTROL FUNCTION OF THE SINUS- GLAND Experiment I This experiment was intended to discover any differences that might occur in the molting process among animals from which both sinusglands had been removed, one sinusgland removed, both sinus- glands removed but with them autoplastically implanted into the ventral abdominal sinus, and finally, completely normal animals. In this experiment four lots of animals were isolated. The first lot of 34 animals was left in perfectly normal condition, though placed in the usual individual glass finger bowls with covers. The second lot of 48 animals was subjected to removal of one eye each. A third lot of 79 animals had both eyestalks removed in the usual manner. The CRUSTACEAN SINUSGLAND AND VIABILITY 111 fourth lot of 44 animals had both eyestalks removed and the contents of their own eyestalks in amphibian Ringer's solution injected into the ventral sinus of the abdomen. Observations were made only with regard to actual molting. The results that were obtained are sum- marized in Table I. TABLE I Data indicating the extent of molting in crayfishes under different experimental conditions. Normal Animals One Eye Off Two Eyes Off Two Eyes Off (Implant) Total no. examined .... No. "molts" 34 9 48 19 79 23 44 3 Per cent "molts" Per cent "molts " dying in process 26 44 40 16 29 74 7 100 Per cent molt/av. life span 2.0 3.4 5.75 1.0 All the records of molting in Table I indicate instances in which the animal either completed the molt or was well along in the process at the time of death. The most significant portion of the table is the item "per cent molt/average life span" which gives the only true figure of the relative rates of molt. The "per cent molts" fail to do this inasmuch as the different lots of animals survived different lengths of time; consequently such animals as normal animals and those with one eyestalk off had a longer time in which molts could occur. On this strictly relative behavior (per cent molt/average life span) the figure for normal animals is 2. With one eye removed, the rate of molt is increased by about 75 per cent, and with the removal of two eyes the molting has been accelerated about 200 per cent. The striking fact, however, is that when both eyes were removed and the eyestalk tissue abdominally implanted, the figure indicating the molt- ing rate is 1, or about half that of normal animals. Were it not for the anomalous molting rate of this last group the results could be interpreted as indicating that the rate of molting is a function of the extent of injury. But, taking the data together, there appears to be a more probable explanation. The eyestalk tissue, under nerve con- trol, liberates a humoral substance into the blood which inhibits the molt. With one eye removed, relatively less substance is liberated and with two eyes removed none of the material, and we see molt correspondingly going on at relatively greater rates. In these terms the explanation of behavior of the last group of animals might be that the implanted glandular tissue continuously liberates some anti- molting substance and the animal is almost unable to molt. 112 F. A. BROWN, JR., AND ONA CUNNINGHAM Some of the acceleration resulting after eyestalk removal may be due to injury effects, but that they are not totally due to injury is indicated by the implantation experiments. Experiment II This experiment points to the sinusgland in the eyestalk as the actual tissue involved in the formation of the molt control humoral substance. The data for this conclusion are taken from observations on molting in the animals in Experiment II on viability. A consideration of the ratio of percentage of completed or nearly completed molts to average survival period, shows that the implan- tation of the sinusgland reduces the molting rate to about one-fifth of that which occurs in the controls with the glandless stalk tissue implants. The conclusions of the former experiment are confirmed and it is further indicated that the sinusgland is the effective tissue in molt control. The results of this experiment are summarized in Table II. TABLE II Data indicating the extent of molting in crayfishes under different experimental conditions. Two Eyes Off Two Eyes Off (Implant) Total no. examined 20 20 No. "molts" 4 1 Per cent molts 20 5 Per cent molts/av. life span 1.57 .31 In the course of this experiment all the animals were carefully watched, not only for completed molts but also for the slightest symp- toms of the beginnings of molt. The early signs of molt were usually indicated by a visible separation between the carapace and the first abdominal tergite. Practically all of the eyestalkless animals, regard- less of the type of implant, showed this separation from three hours to three or four days prior to their death. This was so definite that it was possible to predict the death of any animal within these limits. In many instances this separation was followed by a completed molt, though in the majority of cases the animals died before further steps in the molting process. It is admitted that some other factors, such as change in general tone of the abdominal musculature or upset in the water metabolism of the animal, might be operating in inducing the separation of these two skeletal elements. Superficially, however, we are unable to differentiate between the initiation of the normal molt and its induction by other causes. Furthermore, many of the animals showing this apparent initiation in the molt process showed CRUSTACEAN SINUSGLAND AND VIABILITY 113 muscular activity of the body such as is usually associated with the normal molting process. Those animals from which the eyestalks had been removed and which received the glandless eyestalk implantation, all showed the apparent initiation of molt or completed the molt prior to their death. In three cases the animals completed the molt before death, in one case dying within a day of the molt and in the other cases living two and four days, respectively, after molting. In a fourth case the animal died when well along in the molting process. These facts would indi- cate that even without the eyestalks the animals are physiologically able to complete the molt. But the fact that the eyestalkless animals sometimes continue to live several days after molting and then die without showing further signs of molt, indicates that the sinusgland has a function in addition to molt control. The majority of the animals with sinusgland implants also showed the beginnings of molting prior to their death, just as did the first lot. The only difference between the lots seemed to be that the molting activity was postponed in the case of the implanted animals. These animals seldom do more than show this first sign of molt, scarcely ever proceeding far into the molt or completing it. ' A possible explanation of this is that these animals are prevented from molting by action of the implant until the absence of the eyestalk has worked other degenerating effects upon the organisms to the extent that they no longer have the power to go far with the molt, in spite of removal of the inhibitor through loss of function of the implant. In this regard it would be interesting to trace the rate of degeneration of the im- planted tissue to see if there may be any correspondence between the time of oncome of the molt and the structural degeneration of the implanted cells. It may be possible to interpret the data of Roller (1930) in terms of molt control activity. Animals molting more frequently as a result of absence of a hormone from the sinusgland might well be expected to have less calcium salts in their exoskeleton than normally. SUMMARY 1. Direct evidence for an endocrine activity of the crustacean sinus- gland is given. This evidence has originated from implantation experiments. 2. Removal of the sinusgland significantly shortens the life of the animals, and conversely the length of life of animals with sinusglands removed can be significantly lengthened by implantation of the gland. 3. The sinusgland is readily dissected out in fresh eyestalk tissue 114 F. A. BROWN, JR., AND ONA CUNNINGHAM in strong reflected light. It has a distinctly bluish cast. It is a definite organ which can be readily teased away from the surrounding tissue and removed as a whole. 4. Certain evidence suggests very strongly that a substance con- cerned with the control of molting is elaborated in this gland. The most probable action of this substance is that of inhibiting molt. 5. The action of the sinusgland in molt control appears to be insufficient to explain the viability effect entirely. LITERATURE CITED ABRAMOWITZ, A. A., 1936a. Action of crustacean eye-stalk extract on melanophores of hypophysectomized fishes, amphibians, and reptiles. Proc. Soc. Exp. Biol. and Med., pp. 714-716. ABRAMOWITZ, A. A., 19366. The action of intermedin on crustacean melanophores and of the crustacean hormone on elasmobranch melanophores. Proc. Nat,. Acad. Sci., Washington, 22: 521-523. ABRAMOWITZ, A. A., 1938. The similarity between the hypophyseal chromatophoro- tropic hormone and the chromatophorotropic hormone of the crustacean eyestalk. Physiol. Zool., 11: 299-310. BROWN, FRANK A., JR., 1935. Control of pigment migration within the chromato- phores of Palaemonetes. Jour. Exper. Zool., 71: 1-15. BROWN, FRANK A., 1938. An internal secretion affecting viability in Crustacea. Proc. Nat. Acad. Sci., Washington, 24: 551-555. DARBY, HUGH H., 1938. Moulting in the Crustacean, Crangon armillatus. Anat. Rec., 72: (Suppl.) 78. HANSTROM, B., 1935. Preliminary report on the probable connection between the blood gland and the chromatophore activator in decapod crustaceans. Proc. Nat. Acad. Sci., Washington, 21: 584-585. HANSTROM, B., 1937. Die Sinusdriise und der hormonal bedingte Farbwechsel der Crustaceen. Kungl. Svenska Vetenskap. Handl., Ser. 3, 16 (3): 1-99. HANSTROM, B., 1937. Vermischte Beobachtungen iiber die chromatophoraktivieren- den Substanzen der Augenstiele der Crustaceen und des Kopfes der Insekten. Kungl. Fys. Sdllsk. Handl., 47 (8): 3-11. KLEINHOLZ, L. H., 1938. Studies in the pigmentary system of Crustacea. IV. The unitary versus the multiple hormone hypothesis of control. Biol. Bull., 75: 510-532. ROLLER, G., 1928. Versuche iiber die inkretorischen vorgange beim Garneelen- farbwechsel. Zeitschr.f. vergl. Physiol., 8: 601-612. ROLLER, G., 1930. Weitere Untersuchungen iiber Farbwechsel und Farbwechsel- hormone bei Crangon vulgaris. Zeitschr.f. vergl. Physiol., 12: 632-667. PERKINS, E. B., 1928. Color changes in crustaceans, especially in Palaemonetes. Jour. Exper. Zool., 50: 71-103. STAHL, FILIP, 1938a. Preliminary report on the colour changes and the incretory organs in the heads of some crustaceans. Arkiv.fdr Zoologi, 30B: 1-3. STAHL, FILIP, 19386. Uber das Vorkommen von inkretorischen Organen und Farb- wechselhormonen im Ropf einiger Crustaceen. Kungl. Fys. Sdllsk. Handl., 49 (12): 3-20. WELSH, J. H., 1937. The eyestalk hormone and rate of heart beat in crustaceans. Proc. Nat. Acad. Sci., Washington, 23: 458-460. THE METHOD OF FEEDING OF CHAETOPTERUS G. E. MACGINITIE (From the, William G. Kerckhoff Marine Laboratory of the California Institute of Technology, Corona del Mar, California) INTRODUCTION Ciliated currents present on the surface of animals, when examined under artificial conditions, are seldom, if ever, typical of the animal in its natural environment. Failure to recognize this fact and failure to observe the presence of mucus and note its importance in the feeding process have given rise to many erroneous descriptions of the feeding mechanism of various marine invertebrates. In conformity with the statement made in Science (MacGinitie, 1937), the feeding activities of many marine invertebrates have been investigated (including tuni- cates, pelecypods, gastropods, annelids and coelenterates) , and descrip- tions of the feeding activities of these animals will follow as soon as they can be prepared for publication. This paper will deal with the feeding of the annelid Chaetopterus variopedatus Renier et Claparede. Because of its wide distribution and its usefulness as a source of embryological material, Chaetopterus is well known both abroad and in this country. Also, because of its unusual and somewhat bizarre structure, it has created a great deal of interest from both an anatomi- cal and a natural history point of view (Laffuie, 1890; Enders, 1909). However, no paper that I have seen has given the correct method of feeding of this animal. FEEDING METHOD The structures concerned with the feeding activities of Chaetopterus are the peristomial funnel with its lips, the mouth, the dorsal ciliated groove, which ends in the dorsal cupule of the thirteenth segment, the pair of aliform notopodia of the twelfth segment, and the three fans of the fourteenth, fifteenth and sixteenth segments (see Fig. 1). In preparing to feed, Chaetopterus approaches one or the other end of the leathery U-shaped tube in which it lives and spreads its aliform notopodia out against the sides of the tube. It then begins to secrete mucus from the inner walls of these notopodia, the secretion beginning at the distal ends and proceeding inward toward the body. The cilia of the inner surface of the notopodia carry the mucus across the 115 116 G. E. MACGINITIE opening in a sheet from the distal ends to the body of the worm, whence it is carried posteriorly as a bag by the ciliated groove to the dorsal cupule, where the closed end of the mucous bag is taken into the cup or concave surface of this organ. This creates an elongated bag of mucus, the anterior end of which is fastened to or continuous with the glands lining the inner surface of the aliform notopodia, and the closed posterior end of which is held by and rolled up within the dorsal cupule. A current of water is now maintained through the burrow by the activity of the three fans just posterior to the dorsal cupule. Since vs. FIG. 1. A, Chaetopterus variopedatus within its tube, feeding; B, dorsal surface of anterior portion of worm, a.n., aliform notopodium; c., cirrus;/., fans;/.6., food ball being rolled up within the dorsal cupule; d.g., dorsal ciliated groove; m., mouth; m.b., mucous bag; p.f., peristomial funnel; v.s., ventral suckers. the walls of the burrow are completely in contact with the body of the animal and the aliform notopodia at the anterior end of the mucous bag, it is necessary for the current of water to pass into the bag, out through its sides, and thence along the body of the worm, and ulti- mately to issue from the burrow at th& opposite end. While the current is being maintained by the fans, mucus is continuously secreted at the anterior end of the bag, and, at the same rate, the posterior end is rolled into a ball within the dorsal cupule by the cilia of its inner surface. Since all water entering the burrow while a mucous bag is FEEDING OF CHAETOPTERUS 117 present passes through the walls of the bag, the mucus removes from the current all solid particles, whatever their size. It is these particles which lodge on the inner surface of the mucous bag that constitute the food of Chaetopterus. It consists mainly of detritus (organic debris and bacteria) stirred up from the surface of the ocean or estu- arine bottom by wave action, currents, other animals, etc. Because the entrances to the tube of Chaetopterus are considerably constricted, no very large particles find their way in with the feeding current. Such that do are usually detected by the peristomial cilia of the worm and are passed out at the sides of the worm anterior to the aliform notopodia, which are lifted to allow the material to pass, and so do not find lodgment in the mucous bag. Since the mucus of the bag is being secreted continuously, and at the same time the posterior end is being rolled into a ball in the dorsal cupule, it is evi- dent that the entire bag is constantly being renewed, and that the posterior portion is much more heavily laden with food than is the anterior. When the ball of mucus and food in the dorsal cupule reaches a certain size, the anterior end of the mucous bag is cut off from the notopodia, and the dorsal cupule continues to rotate the ball until the remainder of the bag is completely (or, occasionally, only partly) rolled up. The dorsal cupule is then turned anteriorly and stretched forward somewhat to expel the ball of food onto the posterior end of the dorsal groove. At the same time the action of the cilia of the groove is reversed, and the bolus of mucus with its entrapped food is carried forward along it to the mouth, where the bolus is enveloped by the lips and swallowed. The size of the bolus of food depends upon the size of the dorsal cupule, and, therefore, upon the size of the animal. For a Chaetopterus about 6 inches long the food ball averages about 3 mm. in diameter. When Chaetopterus is feeding there is some variation in the length of its body, particularly in that portion between the head and the dorsal cupule, and, therefore, the length of the mucous bag will vary in the same animal at different times. The following figures are given for a worm 142 mm. in length, measured during a time when the animal was feeding. Fifteen milli- meters posterior to its point of origin, the width of the mucous bag was 6 mm., and the dorso- ventral diameter at the same point was 7 mm. The length of the mucous bag was 37 mm. The rate of secretion of this bag was approximately 1 mm. per second. While the worm was feeding the number of beats for any one of the three fans was 64 per minute, and this rate was the same for this particular 118 G. E. MACGINITIE worm as observed on successive days over a period of several weeks. Although the rate of beating of the fans is quite uniform for any one worm, it varies with individuals, for another worm maintained a rate of 52 beats per minute. From the beginning of the spinning of the mucous bag to the ingestion of the bolus of food required, on the average, 17 minutes, and varied only plus or minus 1 minute from this average. LITERATURE CITED ENDERS, HOWARD EDWIN, 1909. A study of the life history and habits of Chaetop- terus variopedatus, Renier et Claparede. Jour. Morph., 20: 479-531. LAFFUIE, J. J., 1890. Etude monographique du Chetoptere (Chaetopterus vario- pedatus Renier), Arch, de Zool. Exp. et Gen., Ser. 2, 8: 245-360. MACGINITIE, G. E., 1937. The use of mucus by marine plankton feeders. Science, 86: 398-399. THE ACTION OF EYE-STALK EXTRACTS ON RETINAL PIGMENT MIGRATION IN THE CRAYFISH, CAMBARUS BARTON I JOHN H. WELSH (From the Biological Laboratories, Harvard University) I Pigment cells of the retina of the vertebrate eye and pigment cells of the compound eye of arthropods have long been known to lack motor innervation. Hence there has been much speculation regard- ing the nature of the mechanisms controlling the movements of these cells or the pigment within them. As recently as 1932 when Parker reviewed the literature on retinal pigments there was no direct evi- dence as to the nature of the control, but considerable indirect evidence suggested that hormonal agents were responsible for initiating and maintaining retinal pigment migration. The first successful attempt to demonstrate the existence of a hormone acting on the retinal pig- ments of arthropods was made by Kleinholz (1934; 1936). He found that the injection of an active principle from eye-stalks of Palae- monetes into dark-adapted individuals of the same species caused the movement of the distal and reflecting pigments to positions charac- teristic of the light. Studies of the persistence, under constant external conditions, of 24-hour cycles of pigment migration in the compound eye had led to one of the earlier suggestions that there was a hormonal control of retinal pigment (Welsh, 1930; see also Welsh, 1938, for review of the literature pertaining to diurnal rhythms). The extension of these studies to the eye of Cambarus made necessary an investigation of hormone factors in the control of retinal pigment migration in this crustacean. Certain of the results obtained will be presented in this paper. II The majority of observations were made on eyes of Cambarus bartoni but eye-stalks of C. clarkii and C. limosus were sometimes used as sources of the pigment-activating substance. The approximate positions of the three sets of pigment (distal, proximal and reflecting) were determined by briefly illuminating the 119 120 JOHN H. WELSH eye of an animal, in the dark, by a bright beam of light, and observing the amount of light reflected from the eye. This method has been employed by Day (1911). Exact determinations of pigment positions were first made by sectioning the eyes but this is a time-consuming procedure and a rapid method was developed as follows. Animals were killed by dipping in water at 80 C. for 10-15 seconds. The eyes were then removed and split in halves. When these halves were examined under a binocular, using bright reflected light, it was possible to measure the positions of the pigments quite as accurately as in sections. The active substance from the eye-stalk, which may be identical with the chromatophorotropic hormone (Abramowitz and Abramo- witz, 1938), was prepared by grinding 20 eyes and eye-stalks of medium-sized crayfishes in 1 cc. of cold-blooded Ringer, then heating to 100 C. and filtering. The injection of an appropriate volume of the filtrate made it possible to administer the active material from a fraction of an eye-stalk or from one or more eye-stalks as a given dose. Appropriate control injections of Ringer's fluid and of extracts of ventral nerve cord were made and always with negative results. Ill The first experiments to be reported were done to test the effect of the eye-stalk extract on light-adapted eyes. Several C. bartoni were allowed to adapt for several hours in bright diffuse sunlight. At 10:30 A.M. 0.05 cc. of eye-stalk extract (containing the active material from one eye-stalk) was injected in the ventral abdominal musculature of each of half the individuals. At 2:00 P.M. the entire lot was killed with hot water and the eyes removed, split and examined. In Fig. 1 may be seen the distribution of the pigments of a light- adapted eye. The distal pigment forms a sheath around the cone and the process leading from the cone to the rhabdome, but a portion of each distal pigment cell next to the retinular or proximal pigment cells is not filled with pigment. Most of the proximal pigment sur- rounds the rhabdome, but some remains below the basement mem- brane. The reflecting pigment in crayfish eyes does not migrate as it does in some crustaceans (Welsh, 1932). The appearance of an eye of a light-adapted animal, when viewed by reflected light, is shown in Fig. la. The positions of the black screening pigments are such that light cannot reach the reflecting pigment layer nor can light rays, except those which are parallel to the main axis of an ommatidium, reach the rhabdome or light-sensitive element of the eye. Such an eye is called an apposition eye since a given rhabdome receives light RETINAL PIGMENT MIGRATION IN THE CRAYFISH 121 only from its adjacent lens system and is not acted on by light enter- ing at an angle through neighboring ommatidia. -- b". rn. EXPLANATION OF FIGURES con. = cone b.m. = basement membrane d.p.c. = distal pigment cell p. p.c. = proximal pigment cell rh. = rhabdome r.p.c. = reflecting pigment cell FIG. 1. An ommatidium of a typical light-adapted eye showing the positions of the eye pigments. This and the following figures of ommatidia show the situation as seen in thin sections of the eye. In the intact light-adapted eye each cone, cone process and rhabdome is almost completely surrounded by a cylinder of pigment. FIG. la. Showing the appearance of an intact eye with the pigment distribu- tion seen in Fig. 1 when viewed, in the dark, by bright reflected light. FIG. 2. An ommatidium showing the effect on the pigments of injection of eye- stalk extract into a light-adapted animal. FIG. 2b. The intact eye has essentially the same appearance as does the normal light-adapted eye. 122 JOHN H. WELSH An ommatidium from a typical eye of a light-adapted animal, injected with eye-stalk extract and left in the light, is represented by Fig. 2. The distal pigment is in a more extreme proximal position and all of the proximal pigment is above the basement membrane. It is as though the effect of the injected material were added to the effect of light, which probably acts by causing the release of the active material or hormone. The intact eye of such an animal has essen- tially the same appearance as does the normal light-adapted eye (Fig. 2&), although it may not be as black. This is due to the distance of the distal pigment from the surface of the eye. IV When specimens of C. bartoni were placed in the dark, the typical dark-adapted condition in the eye was seen after two hours or less. It is known, however, that there is a diurnal migration of proximal retinal pigment in crayfishes which are kept in continuous darkness (Bennitt, 1932), so in order to assure uniform conditions in all experi- ments on dark-adapted animals the majority of observations were made in the early evening. The positions normally occupied by pigments in a dark-adapted eye are shown in Fig. 3. The distal pigment forms a collar surround- ing the cone and the proximal pigment is all below the basement membrane. In such a condition the rhabdome of a given ommatidium may receive light from neighboring ommatidia. Such an eye is re- ferred to as a superposition eye and is commonly found in those insects and crustaceans which are active at night. The intact eye of a dark-adapted crayfish has a brilliant orange- red center when viewed by reflected light, due to the mirror-like property of the exposed reflecting or tapetal layer (Fig. 3c). The color is due to the visual red of the rhabdomes. When dark-adapted C. bartoni were injected with eye-stalk ex- tracts, and left in the dark, varying effects on the pigment were seen depending on the amount injected, and the interval between the time of injection and the time of observation. Animals which were dark- adapted for several hours and injected in the early evening with an amount of material equivalent to that obtained from one-fourth to one eye-stalk showed, after three hours, a migration of the distal pigment to or toward the light position. The proximal pigment was not affected (Fig. 4). When such eyes are viewed by reflected light they appear gray rather than black and very little light is reflected from the tapetal layer (Fig. 4d). The injection of 0.1 cc. of the extract (= the extractible material from two eye-stalks) had a distinct effect on the proximal as well as RETINAL PIGMENT MIGRATION IN THE CRAYFISH 123 the distal pigment. After three hours both pigments were found to occupy positions more or less typical of light adaptation (Fig. 5). The intact eye when viewed by reflected light had the same appearance as the normal light-adapted eye (cf. Fig. 5e with la). FIG. 3. Ommatidium of a typical dark-adapted eye showing the positions of the pigments. FIG. 3c. The intact eye of a dark-adapted animal has a bright orange-red center when viewed by reflected light. FIG. 4. The injection of the active material from one eye-stalk into a dark- adapted animal causes the migration of the distal pigment to the light position. FIG. 4d. The intact eye of such an animal may have a small reflecting central area. FIG. 5. The injection of the active material from two eye-stalks into a dark- adapted animal causes the migration of both distal and proximal pigments to their light positions. FIG. 5e. The intact eye of such an animal has the same appearance as that of a light-adapted animal. 124 JOHN H. WELSH V It has been demonstrated that it is possible, by means of a simple extraction process, to obtain from two eye-stalks of a crayfish an amount of retinal pigment activator, or hormone, equivalent to that normally released by the animal during the process of light adaptation. From one eye-stalk the amount of hormone is sufficient only to acti- vate the distal pigment cells; thus indicating that they have a lower threshold than do the proximal pigment cells. Such threshold differ- ences between the three sets of pigments in a given species may account for such a situation as was first seen in the eye of Macro- brachium (Welsh, 1930), where under continuous 'illumination the distal pigment cells migrate toward the periphery of the eye at the time of sunset and return to a proximal position at the time of sunrise, while the proximal pigment remains in a constant light position (see also Welsh, 1935, 1936; and Kleinholz, 1937, 1938). The injection of eye-stalk extracts into dark-adapted crayfishes makes it possible to obtain a "light-adapted" eye, as regards the positions of the screening pigments, while the "dark-adapted" level of the light-sensitive substance of the retina remains unaffected. This enables one to study the effect of pigment position on visual acuity and response to flicker and has been employed by Crozier and Wolf (1939). SUMMARY A substance similar to, or identical with, the eye-stalk or chroma- tophorotropic hormone may be obtained from the eye-stalks of cray- fishes. When injected, in proper amount, into light-adapted cray- fishes it causes the distal and proximal pigments to migrate to more extreme "light positions" than normal. When injected into dark- adapted crayfishes which are allowed to remain in the dark it causes the migration of one or both sets of screening pigment to their "light positions." The distal pigment has a lower threshold than the proxi- mal pigment, as it is affected by lower concentrations of the active substance. It is suggested that such threshold differences may ac- count, in part, for the unusual pigment responses which have been observed in compound eyes in studies of 24-hour cycles in pigment migration. LITERATURE CITED ABRAMOWITZ, A. A., AND R. K. ABRAMOWITZ, 1938. On the specificity and related properties of the crustacean chromatophorotropic hormone. Biol. Bull., 74: 278. BENNITT, R., 1932. Diurnal rhythm in the proximal pigment cells of the crayfish retina. Physiol. Zool., 5: 65. RETINAL PIGMENT MIGRATION IN THE CRAYFISH 125 CROZIER, W. J., AND E. WOLF, 1939. The flicker-response contour for the crayfish. II. Biol. Bull., 77: \26. DAY, E. C., 191 1. The effect of colored light on pigment-migration in the eye of the crayfish. Bull. Mus. Comp. Zoo!., 53: 305. KLEINHOLZ, L. H., 1934. Eye-stalk hormone and the movement of distal retinal pigment in Palaemonetes. Proc. Nat. Acad. Sci., 20: 659. KLEINHOLZ, L. H., 1936. Crustacean eye-stalk hormone and retinal pigment migra- tion. Biol. Bull, 70: 159. KLEINHOLZ, L. H., 1937. Studies in the pigmentary system of Crustacea. II. Diurnal movements of the retinal pigments of Bermudan decapods. Biol. Bull., 72: 176. KLEINHOLZ, L. H., 1938. Studies in the pigmentary system of Crustacea. IV. The unitary versus the multiple hormone theory of control. Biol. Bull., 75: 510. PARKER, G. H., 1932. The movements of the retinal pigment. Ergbn. der Biol., 9: 239. WELSH, J. H., 1930. Diurnal rhythm of the distal pigment cells in the eyes of certain crustaceans. Proc. Nat. Acad. Sci., 16: 386. WELSH, J. H., 1932. The nature and movement of the reflecting pigment in the eyes of crustaceans. Jour. Exper. Zool., 62: 173. WELSH, J. H., 1935. Further evidence of a diurnal rhythm in the movement of pigment cells in eyes of crustaceans. Biol. Bull., 68: 247. WELSH, J. H., 1936. Diurnal movements of the eye pigments of Anchistioides. Biol. Bull., 70: 217. WELSH, J. H., 1938. Diurnal rhythms. Quart. Rev. Biol., 13: 123. THE FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH II. RETINAL PIGMENT AND THE THEORY OF THE ASYMMETRY OF THE CURVE W. J. CROZIER AND ERNST WOLF (From the Biological Laboratories, Harvard University, Cambridge) I The flicker-response contour (F - log /) for the crayfish Cambarus bartoni resembles that for other arthropods having markedly convex eyes (see Crozier and Wolf, in press). Only its very uppermost part can be fitted by a probability integral. Over its lower part the slope increases too rapidly, so that the whole curve is quite asymmetrical. This departure from the rule observed in the responses of vertebrates (see Crozier and Wolf, 1937a and b, 1938a) has been accounted for (Crozier and Wolf, 1937 c, 19386) by the shape of the optic surface in the majority of arthropods. With increasing flash-intensities the retinal area effectively involved is increased, which results in a higher F; this is due to the greater chance of exciting ommatidia toward the circumference of the curved eye. Confirmation of this view, consistent with the consequences of changing the light-time fraction in the flash- cycle (Crozier and Wolf, 1937c, 19386), is given by the fact that an arthropod with sufficiently flat optic surfaces, the isopod Asellus (Crozier and Wolf, 1939), gives a flicker-response contour which is a perfectly symmetrical probability integral. The asymmetry of the curve with Anax is appropriately reduced by blocking out all but a central area of the eye (Crozier and Wolf, 1937c, 19386), and in a form with still more markedly curved optic surfaces (Cambarus) (see Crozier and Wolf, in press) the asymmetry is much more extreme. In our experiments with Anax (Crozier and Wolf, 1937c, 19386) the limitation of the increase of effective retinal area with increase of illumination by painting portions of the eyes was recognized to be im- perfect. A certain amount of leakage of light near the margins of a cap of enamel, and under its edge, cannot be prevented. A neater method of accomplishing the purpose is to use the migrations of retinal pigment cells. The flicker-response contours we have discussed were determined with animals previously dark-adapted. For such a crusta- cean as Cambarus this means that the proximal retinal pigment is below the level of the receptive retinulae, the distal pigment cells well 126 FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH 127 out toward the surface of the eye around the crystalline cones. The retinulae are completely unshielded from laterally spreading light, and the condition is that for the "superposition " type of eye (Exner, 1891). In the eye well light-adapted the forward migration of the proximal pigment shields the retinulae, while the inward movement of the distal pigment forms around each ommatidium an opaque tube of pigment along the length of the crystalline lens and down to the proximate pigment (Bernhards, 1916; Day, 1911; Parker, 1932). The effective isolation of each recipient unit from light other than that proceeding down the axis of the ommatidium then produces the condition for the "apposition eye" (Exner, 1891). For our purposes, however, no use could very well be made of the control of retinal pigment migration by light. The process of light adaptation involves not only movements of the retinal pigment cells, but also, it must be presumed, the intrinsic photic adaption of the visual response system itself. At the same time, if some other pro- cedure could be found to cause the retinal melanophores to assume the ' "light-adapted" condition, it should serve admirably for a test of certain properties of the Cambarus flicker-contour. It should also give some direct behavioral evidence as to the functional role of the retinal pigment and its movements, as well as providing material for a logical approach to the method of estimating the time-course of visual light-and-dark-adaptation in such animals. It was pointed out to us by Dr. J. H. Welsh that extracts containing the "eyestalk hormone" from the optic peduncle produce an effect on the melanophores and also on the movement of retinal pigment (Kleinholz, 1934, 1936, 1938; Welsh, 1939) in dark-adapted eyes of Cambarus, so that injection of sufficient extract into a dark-adapted animal leads to the migration of retinal pigment into positions charac- teristic of the normal light-adapted state. This we have verified in C. bartoni. II The observational procedure was identical with that employed in measuring the flicker-response contour for dark-adapted Cambarus (Crozier and Wolf, in press): temperature 21.5, 50 per cent light-time in the flash cycle. To keep the handling of the animals uniform with respect to time after injection and the like, a lot of 5 rather than of 10 was used. The eyestalks from 10 Cambarus bartoni were extracted in Ringer solution. Into each crayfish prepared for observation there was injected into the abdomen 0.08 ml. of extract, the equivalent of 2 eyestalks. After 75 to 90 minutes in the dark the crayfish are 128 \V. J. CROZIER AND ERNST WOLF bluish in body color and by means of a beam of light directed into the eye the retinal pigment is seen to be in the position characteristic of light adaptation. Sectioned eyestalks fixed in hot water at this stage show the condition clearly under the ultrapak microscope. In the normal dark-adapted eye the proximal pigment is retracted below the basement membrane, while the distal pigment is out between the crystal cones. There is no detectable pigment between the om- matidial units. After about 90 minutes in darkness subsequent to injection of eye-stalk extract, the proximal pigment surrounds the TABLE I Data for the flicker-response contour of the crayfish Cambanis bartoni, with eye-pigment in the "light adapted " state as result of injection of eye-stalk hormone. N = 5 individuals, n 3 observations on each; the same individuals used through- out; t = 21.5 C.; IL = to. See Fig. 1. 7 in millilamberts, F in flashes per second. P.E.i = P.E. of the dispersions. F F m P.E. 1Fl logl m log P..i/! 2 3.3404 5.9958 5 2.0777 5.3334 8 2.2584 5.4806 12 2.5249 3.8155 16 2.6674 5.9737 20 2.7771 3.0398 25 2.9254 3.8291 30 1.0730 3.2355 35 1.2865 3.6169 40 1.5937 3.8785 41.79 0.289 0.00 42 0.0233 2.3664 43 1.0077 1.2823 43.10 0.357 0.50 43.23 0.361 1.00 43.66 0.282 1.25 44 1.5231 0.1464 44.05 0.130 1.50 retinulae, while the distal pigment now envelopes each ommatidial unit down to its base. The condition is one of quite complete shielding of each ommatidium by a dense layer of black pigment, more extreme than is the case in ordinary light adaptation. Ill The determinations of mean critical flash-intensity and mean criti- cal flash-frequency for response (Crozier and Wolf, in press) to visual flicker are given in Table I. Comparison with the results for normally FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH 129 dark-adapted Cambarus bartoni (Crozier and Wolf, in press) shows that there is a pronounced (reversible) effect of the injection of eyestalk hormone upon the properties of the flicker-response contour. This cannot reasonably be traced to an effect of the eye-stalk extract upon the intrinsic processes of photic excitability, for several reasons. In normal with E.S.E FIG. 1. The variation of I\ for normal Cambarus bartoni and after injection with eye-stalk extract (E.S.E.); Table I; see text. the first place injection of ca. 0.06 ml. of the eyestalk extract into A nax (dragon fly) nymphs produces no detectable effect either on pigment migration or on the flicker-response curve, as the following observations showed (tests on 5 individuals) : F 20 30 Normal log /, 2.473 2.749 2.741 Normal + eye- stalk extract log /m 2.478 2.745 2.750 Any effect of this sort would thus have to be specific. In the second place, the results of adapting Cambarus are rapidly apparent even when the retinal pigment is already fully advanced into the "light" position, as subsequently shown ( IV). Finally the various modifica- 130 W. J. CROZIER AND ERNST WOLF tions of the flicker-response contour are those to be expected as the result of the optical shielding of the ommatidia, so that no specific effect on excitability need be invoked. For any given level of flash-intensity the variation of I\ among the individuals used is statistically of the same magnitude as for the normal group previously examined (Crozier and Wolf, in press). The 5 indi- viduals giving the data of Table I were in the lot of 10 providing the normal curve for this species (Crozier and Wolf, in press). The scatter of the variation indices (P.E.iJ is even a little less than might have been expected in view of the smaller number of readings in the eyestalk injection series (Fig. 1). The effects to be expected if the "dark" position of the retinal pigment shields ommatidia from all but light parallel to the retinular axis, and if this is to prevent the recruitment of optic impulses from a larger retinal surface as flash intensity is increased, are the following: (1) the total achievable sensory effect (= F max .} must be reduced; (2) at given /, F must be less; (3) the asymmetry of the F - - log / curve must be markedly reduced ; and (4) it would not be surprising to find the slope of the "fundamental " curve increased (i.e., a'\ os /, for the ideal frequency distribution of log / thresholds, reduced), owing to the mechanical exclusion of a large proportion of the otherwise marginal!} excitable units. FIG. 2. F log / curves for dark-adapted Cambarus and under the same conditions for individuals injected with eye-stalk extract (E.S.E.); Table I. Figure 2 shows that the F - - log / curve with Cambarus dark- adapted but under the influence of eye-stalk extract is moved toward higher intensities and exhibits a lower maximum. These are the FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH 131 100 80 60 Cambarus normal 20 FIG. 3. The curves of Fig. 2 brought to the same F mil *. (= 100 per cent), to show change of shape. results of a decrease in the total number of excitable elements (Crozier and Wolf, 1937c, 19386), as expected. The asymmetry of the curve is also decreased (Fig. 3). The 50 40 2,0 10 FIG. 4. The curves of Fig. 2 with probability integrals adjusted to the upper portions (cf. Crozier and Wolf, 1937f, 1938&, 1939, and paper in press), to show that the flicker-response contour after injection of eye-stalk extract departs less than the normal; see text. 132 W. J. CROZIER AND ERNST WOLF sheathing of the ommatidia by pigment materially reduces the chance of photic action on additional elements as intensity increases, hence the slope of the F - - log / curve cannot increase so rapidly. It is to be presumed that in the absence of comparatively free passage of light through the eye (as in the dark-adapted state), the actual intensity at each receptor locus will be decreased. This cannot be a major factor in the changes shown in Fig. 2, else increase of in- tensity would find the F - - log / curve continuously rising at its upper end. The diffusion of light within the substance of the eye cannot be ignored, however. Figure 4 shows that the asymmetry of the flicker- FIG. 5. Portions of the curves in Fig. 2 for dark-adapted Cambarus with (D + E.S.E.) and without (D) injection of eyestalk extract, and the first (1) and second (?) sets of readings (Table II) during the progress of dark adaptation after light adaptation. response contour has been decreased (cf. Fig. 3), but not abolished. In view of the proximal movement of the distal retinal pigment under the influence of eye-stalk extract (Kleinholz, 1934, 1936, 1938; Welsh, 1939), this is not surprising. It probably explains the slight but detectable rise of the curve at the highest intensities used (Table I ; Fig. 2), particularly when F m is determined at constant flash-intensity; this cannot be accounted for by light adaptation ( IV). With allowance for this effect, a reasonable adjustment of an ideal probability integral can be made to the upper part of the curve (Fig. 4). FLICKER-RESPONSE CONTOUR FOR THE CRAYFISH 133 Comparison with the normal, in the same figure, shows that - S o .9 o rt b/l eu 4J bfl o _c c _rt C en 13 '5 4-1 C 6 s. i o en l^ 4= GJ ^ eu en +- 1 3 1 '5 "o i^ en O *^_ *j c C 4- i^ O 3 C o eu u O *- en eu 15 " (J 2f 2 eu rt-f v-> 1 ' r- (U -C K j> en -M '"-f cj -C > ' CX O en M 2 C a. c cfl rt .2 4J ^ 10 I en eu 8 rt s ^5 a c ^ "i5 M ^ o 6 o 4-< O 2 e a Js o ABSENCE OF EPITHELIAL HYPOPHYSIS 181 e 03 O C tfi 03 -o a 3 03 4) .22 u -0 ^ ^ 43 < J 5 ^ W \1 fcuO en C -^. .s , O 6 L Q_ > T3 -o i c M c^v^- ? ~o _t a_ T? E 00 I a- . 4) ' ' 4^ - l_ 43 CO u. 4) "-> 43 03 f~* . . . O fj -u o (D 4) = ^^? 1 ^' < o ~ 4) 03 4) OJ "^ C o s - .2 4-" O -S S3 tfi Cy O ^^ ^-^ 3 c r rt 43 ~ <= 43 -4_* rl-M ^ E .-tt O ^ 03 r- C *-*- S'o.o o E CO r^ (-H , % ,j 1 w o oj c.2 2 '-g " 8 S 8* = " bfl 41 in ft 03 bo' O o bO 43 a C fj O 4^ 01 bo 03 03 oo" "g O o-a QJ ^* . 43 . a o -M o '5 4) 'in 03 182 DON WAYNE FAWCETT Furthermore, the interdependence in development of one part of the pituitary upon another may be quite different in mammals than in amphibia and fishes. The present case of pituitary agenesis in a lower vertebrate seems to bear out Smith's observations, for here, as in his tadpoles, in the total absence of the buccal ectoderm, the pars neuralis has not attained its normal shape nor has it undergone its typical differentiation into a saccus vasculosus. Tadpoles in which the buccal hypophysis has been removed at an early stage of development display albinism in which the epidermal melanophores are diminished in number and pigment content besides remaining in a persistent state of contraction. These conditions in the tadpole are closely paralleled by this albino dogfish fetus in which the melanophores are less numerous, contracted, and noticeably with- drawn from the surface. Lundstrom and Bard, in a study of the effects of ablation of various parts of the brain of the dogfish (Mustelis canis}, first discovered the hypophyseal control of the cutaneous pigmentation in the elasmobranch fishes. They found that removal of the neuro-intermediate lobe invariably resulted in pallor of the skin. The present anomalous specimen constitutes an interesting confirma- tion of their work. Evidently there has been a spontaneous suppres- sion of the oral hypophysis equivalent to actual experimental ablation. Because of the intermingling of the elements of the pars neuralis and pars intermedia of the dogfish pituitary, it has so far been impossible to accomplish a complete operative separation of these two parts. The presence of the neuro-hypophysis in the present specimen, but the total absence of the oral pituitary (including the pars intermedia) indicates that the humoral agent affecting pigmentation is a derivative of the oral components of the gland. Presumably in that portion of the gland referred to as the neuro-intermediate lobe it is the buccal elements that are responsible for the chromatophore-expanding factor. Observations in many cases of human anencephaly (Covell, 1927) make it apparent that aberrant formative processes involving defective closure in the dorsal midline may result in agenesis of the neural lobe of the pituitary. Cyclopia and astomia are occasionally found together in human monsters. It appears from the present observa- tions that, in the dogfish, anomalous development in the ventral mid- line with imperfect separation of symmetrical parts and consequent cyclopia and astomia may result in agenesis of the oral hypophysis. ABSENCE OF EPITHELIAL HYPOPHYSIS 183 SUMMARY 1. An anomalous fetus of the spiny dogfish (Sqiialus acanthias) is described in which there are malformations of the head comprising cyclopia, astomia, and abnormalities of the hypophysis. 2. The abnormalities of the hyophysis involve: (a) The total absence of the oral components of the gland, and (b) A neural lobe which is deformed and possesses no saccus vasculosus. The conclusion is drawn that the neural lobe has not undergone normal differentiation because it has been deprived of its usual association with the buccal hypophysis. 3. The specimen is albino, displaying a diminished number of chromatophores in a state of persistent contraction. This finding indicates that the melanophore-controlling principle in the dogfish is a derivative of the buccal components of the pituitary. 4. Only one other instance of total spontaneous suppression of the oral hypophysis is described in the literature, namely, in a pig fetus (Holt, 1921). In human fetuses anencephaly occurs not infrequently but is associated with suppression of the neuro-hypophysis instead of with the adenohypophysis. BIBLIOGRAPHY BUTCHER, E. O., 1936. Histology of the pituitaries of several fish. Bull. Mt. Desert Island Biol. Lab., pp. 18-20. COVELL, W. P., 1927. A quantitative study of the hypophysis of the human an- encephalic fetus. Am. Jour. Path., 3: 17-28. HOLT, E., 1921. Absence of the pars buccalis of the hypophysis in a 40-mm. pig. Anat. Rec., 22: 207-216. LUNDSTROM, H., AND P. BARD, 1932. Hypophyseal control of cutaneous pigmenta- tion in an elasmobranch fish. Biol. Bull., 62: 1-9. SMITH, P. E., 1920. The pigmentary, growth, and endocrine disturbances induced in the anuran tadpole by the early ablation of the pars buccalis of the hypophysis. Am. Anat. Mem., No. 11, pp. 5-151. VARIATIONS OF COLOR PATTERN IN HYBRIDS OF THE GOLDFISH, CARASSIUS AURATUS H. B. GOODRICH AND PRISCILLA L. ANDERSON 1 (From the Department of Biology, Wesleyan University] This paper gives an account not only of the differences between fish arising from the same genetic cross but also of the variations of color pattern taking place during the life of individual fish. The cross between the common goldfish and the transparent shubunkin which are both varieties of the species Carassius auratus was first subjected to genetic analysis by Berndt (1925 and 1928) and Chen (1925 and 1928). The results indicated that the two parental types are genetically distinguishable by a single gene difference. The formulae as denoted by Chen are : common goldfish TT, the transparent shubunkin T'T', and the hybrid TT'. This hybrid is known to the fanciers as the calico shubunkin. The common goldfish, which is quite brown or black during youth, changes to the familiar orange or red type by destruction of part or nearly all of its melanophores (Berndt, 1925; Goodrich and Hansen, 1931). This type also carries at least two layers of reflecting tissue, one beneath the scale layer and the other backing each individual scale. The transparent shubunkin has lost most of the chromatophores (both melanophores and xantho- phores) and also most of the reflecting tissue. The heterozygous type, or calico fish, shows great variability in the distribution of both melano- phores and xanthophores and there is no bilateral symmetry of pattern. A deep abdominal layer of reflecting tissue is present and a few scales are also backed with the tissue. For full details, papers by Chen (1928) or Goodrich and Hansen (1931) may be consulted. Goodrich and Hansen (1931) made a detailed comparative study of the history of the melanophores of the three phenotypes covering the first eight weeks after hatching during which period the fish grew from 4.5 mm. to about 33 mm. in length. It was found that the history of the three types was similar for the first week (to 9 mm.) showing a uniform rate of multiplication of the chromatophores. After this the three types diverged. The normal goldfish showed a very rapid 1 This paper is published as part of a research program at Wesleyan University supported by the Denison Foundation for Biological Research. The authors wish to acknowledge their indebtedness to Miss Marian Hedenburg for carrying on the program during the last half-year. 184 VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 185 and uniform increase in number of chromatophores. In the trans- parent shubunkin the melanophores began to disintegrate until nearly all were destroyed. The hybrid, however, was found to be highly variable, showing great diversity between individuals. New cells appeared; others were destroyed. It gave the impression of a conflict between the cell proliferation and cell destruction. MATERIAL AND METHODS This paper continues the observations on the melanophore pattern of the heterozygous type beginning where the previous study was discontinued. The work was begun during the summer of 1937 with fish varying from 23 to 36 mm. in length (tip of mouth to base of caudal fin). The fish were chiefly obtained from the Grassyfork Fisheries of Martinsville, Indiana, to which institution we are greatly indebted. The hybrid fish were obtained directly from the hatchery which raises them regularly for the market. Records were made by photographing one side of the fish at intervals of approximately one month, but the periods were lengthened to longer intervals during the last six months. Ten of the fish are still under observation at this time, one year and six months after the start of the work. They vary from 47 to 58 mm. in length. All others that were started died. Anaesthetization, necessary for photography, proves to be fatal in some cases. The individuals differ markedly from each other. For purposes of description tw r o types, A and B, may be recognized, but it should be understood that there are intermediate gradations. Type A shows a relatively uniform distribution of melanophores on the dorsal half of the body and extending variably below the lateral line (Figs. 1 and 2). In Type B, the distribution of melanophores is much more uneven. They tend to be aggregated in clusters (Figs. 4 and 5). Xanthophores are present in both types and are unevenly distributed, but are not studied in this paper as it is very difficult to distinguish and identify the individual cells. HISTORY OF COLOR PATTERNS Type A It is possible with these fish to enumerate and reidentify from time to time all cells of large areas on the photographed side of the body. Except in the cases where wholesale destruction of melanophores occurs, it is found that few cells are lost and that individual cells have long life. An example may be taken (our fish number MG-3) on which 907 cells were enumerated and located on the side of the body (see 186 H. B. GOODRICH AND PRISCILLA L. ANDERSON Table I for this and other references to cell counts). The first photo- graph was taken August 2, 1937 and the last February 17, 1939 making a total series of 18 photographs. During this time, 50 of the 907 cells disappeared and three new ones appeared. Figure 1 is the photograph TABLE I This table gives records of photographs of four of the fish studied. The dates are accurate only for MG-3 as it was not always possible to take all photographs on the same day. MG-3 MG-4 MG-IS MG-16 Calico Type A Calico Type B Transparent Calico Type .4 907 cells 97 cells, 2 cl. 21 cells 613 cells D A D A D A D A July 17, 1937 0(4) Aug. 2 0(1) 9 1 cl Aug. 26 1 6 2 6 1 cl Sept. 20 5 2 1 Oct. 18 5 1 1 cl Nov. 16 5 7 1 (6) Dec. 15 4 4 1 cl Jan. 26, 1938 2 3 4cl Feb. 23 4 7 2cl Mar. 28 1 1 3cl May 2 4 1 1 1 June 7 9 1 1 1 (7) July 16 2 1 Aug. 5 2 1 Sept 13 - - 8 Oct. 12 4 1 Nov 14 Jan. 4, 1939 0(2) 0(5) Feb. 17 2 Totals 50 3 51 2 4 All 1 cl 12 cl D number of cells that disappeared since preceding photograph. number of new cells appearing since preceding photograph, cl cell cluster or spot. (1). (2), (4), (5) indicate pictures reproduced in Figs. 1, 2, 4, and 5. (6) time of beginning of wholesale destruction of melanophores. (7) time at which all melanophores were destroyed. taken August 2, 1937 and Fig. 2 that of January 4, 1939. The dotted lines outline arbitrarily delimited areas marked on the prints to facili- tate the counting and identification of cells. The small circles indicate the former location of cells that have disappeared. Figure 2 is taken at a lower magnification than Fig. 1 and fish had grown from 26 mm. VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 187 to 58 mm. in length (exclusive of caudal fin). Figure 3, however, shows the rectangular area of Fig. 2 raised to the same magnification as Fig. 1. - i * If 1 :* * * *. 9 . , I * * FIG. 1. Fish MG-3. Photograph taken August 2, 1937. X5} 2 - This is a "Type A" calico shubunkin; 907 cells are located in outlined areas. FIG. 2. Fish MG-3. Photograph taken January 4, 1939. X 1 1 A. Fifty-two cells have been lost and 3 new cells appeared since record of Fig. 1 . Dotted circles indicate location of cells that have disappeared. FIG. 3. Section outlined by dashes in Fig. 2 enlarged to same magnification as in Fig. 1, showing increase in size of area and of cells. 188 H. B. GOODRICH AND PRISCILLA L. ANDERSON TypeB These fish show the irregular mottling which is prized by the fanciers. The dark spots are usually clusters of small melanophores too densely crowded to count. Of 97 selected on the first photograph of MG-4 on July 18, 1937, 46 remained on January 1, 1939 (Figs. 4 and 5). In the meanwhile, however, others have appeared and ff * ^ FIG. 4. Fish MG-4. Photograph taken July 18, 1937. X FIG. 5. JlfG-4. Photograph taken January 4, 1939. X2*2. Fifty-one cells disappeared and 12 new cell clusters appeared in area under observation. there has been a notable eruption of spots, or clusters of melanophores 12 altogether on the left side. These spots are first recognized as one or a few minute melanophores which rapidly increase in number. A spot for a time is often bounded by the posterior edge of a scale. Indeterminate Types In many cases the clusters of small cells appear among, or super- ficial to, cells uniformly distributed and in this way combine character- VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 189 istics of Types A and B. An example is MG-5, where it was possible to identify cells only in a small area. Ten disappeared out of 164 in this area, but six new clusters of cells have arisen similar to those discussed under Type B. Extensive Cell Destruction Occasionally a sweeping destruction of melanophores occurs within a few weeks. This is similar to the process in the ordinary goldfish, which is gold because melanophores but not xanthophores have been destroyed. This change most frequently takes place in ordinary goldfish at about three months of age but may occur much later (Cf. Berndt, 1925; Chen, 1925; and Goodrich and Hansen, 1931). It occurred in two of the calico shubunkins which we had under observa- tion in this series. The history of one of these, MG-16, is given in Table I and in this the breakdown occurred at about eight months of age. INCREASE IN SIZE OF CELLS As mentioned above, Fig. 3 shows the rectangular area marked on Fig. 2 enlarged to the same magnification as Fig. 1. The compari- son of Figs. 1 and 3 then shows the actual increase in size of the area outlined. It also shows that the individual cells, which for the most part show approximately the same degree of melanin dispersion in both pictures, have definitely increased in size. It, therefore, appears that, in so far as the melanophores are concerned, the increase of body size has involved an enlargement of cells rather than a multiplication of cells. DISCUSSION These observations not only show that there is much variation among individuals of these hybrids but also that each individual is variable in respect to color patterns displayed during its life cycle. The heterozygous type, as noted for earlier developmental stage by Goodrich and Hansen (1931), continues in later stages to be in a con- dition of unstable equilibrium between opposing tendencies those of cell multiplication and cell destruction. Fukui (1927 and 1930) has shown that the destruction of melano- phores in the ordinary goldfish tends to take place in definitely bounded areas, giving rise to some degree of uniformity of pattern in black- and goldfish. These areas, he believes, correspond to regions of looser subcutaneous tissue bounded by more dense tissue. In effect, these may be perhaps regarded as sinuses filled with tissue fluids or lymph. 190 H. B. GOODRICH AND PRISCILLA L. ANDERSON His experiments with injection of adrenalin showed a restoration of pigment which tended to be circumscribed in such areas. These results suggest that endocrine factors operating on such a region bring about under certain conditions the destruction of chromatophores and under other conditions the production of pigment. Fukui suggests that pigment destruction is due to a higher metabolic rate in these areas, but this might be stimulated by the chemical environment. In contrast to the above, the origin of new spots or cell clusters is entirely irregular, having no relation to the areas described by Fukui. It, therefore, seems unlikely that their location can be due to endocrinal conditions. It then seems probable that the goldfish presents a new example of the dual gene control such as has been suggested in the plum- age of birds. In the case here described the direct gene action may control cell multiplication, resulting in the formation of cell clusters or spots, while remote gene control of "endocrinal regulation" may cause the destruction of cells (see Danforth, 1932, p. 33). A discussion of the developmental origin of cell clusters will be presented in the companion paper, Goodrich and Trinkaus (p. 188). SUMMARY 1. The FI heterozygous types from the cross of the common goldfish with the transparent shubunkin (both of the species Carassius auratus) show not only a great range of variability between individuals, but frequently the pattern of a single individual changes markedly during the life cycle. This is due to destruction and emergence of chromato- phores producing a varying pattern. It is suggested that the multipli- cation of cells is an example of "direct gene control" and the destruc- tion is due to "endocrinal regulation" or remote gene control. 2. Many individual melanophores are long-lived, having been iden- tified at the beginning and end of the 19-month period of observation. 3. Such long-lived melanophores gradually increase in size during the growth of the fish. BIBLIOGRAPHY BERNDT, WILHELM, 1925. Verererbungstudien an Goldfischrassen. Zeitsclir. f. Indukt. Abst. u. Vererb., 36: 161-349. BERNDT, WILHELM, 1928. Wildform und Zierrassen bei der Karausche. Zool. Jahrb., Abt. Allgem. Zool. u. Physiol., 45: 841-972. CHEN, SHISAN C., 1925. Variation in the external characters of goldfish, Carassius auratus. Contr. Biol. Lab. Sci. Soc. China., 1: 1-65. CHEN, SHISAN C., 1928. Transparency and mottling, a case of mendelian inheritance in the goldfish, Carassius auratus. Genetics, 13: 434-452. DANFORTH, C. H., 1932. In Allen, E. Sex and Internal Secretions, pp. 12-54. Baltimore. VARIATIONS OF COLOR PATTERN IN HYBRID GOLDFISH 191 FUKUI, KEN'ICHI, 1927. On the color pattern produced by various agents in the goldfish. Folia Anal. Japan., 5: 257-302. FUKUI, KEN'ICHI, 1930. The definite localization of the color pattern in the goldfish. Folia Anat. Japan., 8: 283-312. GOODRICH, H. B., AND I. B. HANSEN, 1931. The postembryonic development of mendelian characters in the goldfish, Carassius auratus. Jour. Exper. Zool., 59: 337-358. GOODRICH, H. B., AND J. P. TRINKAUS, 1939. The differential effect of radiations on mendelian phenotypes of the goldfish, Carassius auratus. Biol. Bull., 77: 188-195. THE DIFFERENTIAL EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES OF THE GOLD- FISH, CARASSIUS AURATUS 1 H. B. GOODRICH AND J. P. TRINKAUS (From the Department of Biology, Wesley an University) The types of goldfish used in the following experiments are those described in the companion paper by Goodrich and Anderson (1939). These are the common goldfish, the transparent shubunkin, and the hybrid between these two known as the calico shubunkin. Genetic analysis has shown that this is a monohybrid cross and the formulae assigned have been: ordinary goldfish TT, the transparent shubunkin FT', and the calico fish TT'. The original purpose of the ultraviolet treatment was to destroy certain parts of the color pattern in the calico fish and to study its regeneration. It was, however, soon discovered that lighter treatment than that needed to destroy the chromatophores apparently induced the formation of new pigmented areas. Consequently a more careful program of experimentation was outlined to verify these preliminary findings. METHODS The source of illumination has been a small laboratory mercury lamp obtained from the Hanovia Company (their model E). The quartz tube is 16 mm. in diameter, has a length of arc of 50 mm., and operates on 110-volt circuit. For purposes of destruction of melano- phores, treatments frequently of 30 minutes or more were administered, but for stimulation of pigment formation most treatments were of 10 minutes duration at distances varying from 2 cm. to 6 cm. from the lamp. Only a small area was irradiated on each fish. Other parts of the body within the zone of illumination were protected. The areas treated varied from about 0.2 to 0.9 sq. cm. in size. These were delimited by pieces of wet filter paper over which were placed pieces of tin foil, which in turn were held in place by more filter paper. Wet cotton was put over the head and the operculum and over the rear of 1 This paper is published as part of a research program at Wesleyan University supported by the Denison Foundation for Biological Research. The authors wish to acknowledge their indebtedness to Miss Priscilla Anderson who performed the preliminary experiments. 192 EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 193 the body and caudal fin. This kept the fish moist and helped to hold it in place. The fishes were anesthetized in a 1 per cent urethane solution and were placed on a paraffin block modeled to hold the fishes nearly upright. During irradiation the spot treated was kept wet with distilled water to prevent drying of the tissue. Photographs of both sides of the fish were taken before treatment. The irradiated areas were outlined on the photographic prints and later the location of new spots was marked on these prints, or additional photographs taken if thought desirable. The fish were inspected at weekly intervals for the first three months after the treatment and those fishes that survived were observed at longer intervals for the succeeding six months. EXPERIMENTS After the preliminary experiments, it was first planned to treat approximately equal numbers of the three Mendelian types. Ac- cordingly, ten of each were irradiated. Later, the numbers treated were increased, especially of the hybrid type which was the only form which gave a positive reaction. The final lot of fish irradiated included 24 of the ordinary goldfish TT, 17 of the transparent shubunkin T'T', and 52 of the hybrids TT', giving a total of 93 fish treated. 2 Areas with few or no melanophores were selected for irradiation. The essential result from the comparative study was that the hybrids alone showed a positive reaction by development of new melanophores, while in the two parental types no melanophores were formed. Most fish in all these groups exhibited inflammation and sometimes necrosis of tissues. In the goldfish TT the xanthophores and guanin crystals (of the reflecting tissue) were frequently destroyed. Spots or cell clusters appeared only in the hybrids. These were first observed as small faintly grayish chromatophores, having long delicate processes. The number of cells increased and in about eight weeks these cells became typical mature goldfish melanophores. (Figure 3 shows the inflammation following irradiation, and Figs. 4 and 5 the development of a cell cluster in the same spot.) Figure 1 is a photograph of a hybrid TT' taken on March 29 just before radiation and the area irradiated is outlined. Figure 2 is of the same fish on May 27. Three new spots, one small and two large, have appeared in the radiated area and one outside (in dotted circle). All but one of the new spots 2 Eight fish of doubtful classification are excluded from these totals. Inspection of pattern indicated that they probably were one normal goldfish and seven trans- parent shubunkins. All gave negative reactions. Even if the presumed transparent types were transferred to the list of 52 calico shubunkins the essential results as indicated by the graphs, Figs. 6 and 7, would not be altered. 194 H. B. GOODRICH AND J. P. TRINKAUS were located in the dermis superficial to the scales. This one excep- tional spot was beneath the scales. The companion paper (Goodrich and Anderson, 1939) has shown that the hybrid or calico fish is characterized by an irregular mottling and, moreover, that this pattern is subject to change during the life of the individual. It therefore seemed possible that the appearance of new spots after radiation might be nothing more than the normal sequence of events. On this account, many more of the hybrids were -**T' v FIG. 1. Photograph of a hybrid TT' taken on March 29 just before radiation- The area later irradiated is outlined with dotted line. X 1/4- FIG. 2. Photograph of same fish as in Fig. 1 taken on May 27. Three new spots (one small and two large) have appeared in the radiated area and one outside (in dotted circle). X 1J4- FIGS. 3, 4, 5. Successive photographs of the same area on a hybrid fish. X 6. Irradiation Nov. 13, 1937. Fig. 3, appearance Nov. 27; congestion of capillaries in center (an older spot at right). Fig. 4, Jan. 2, 1938. Fig. 5, Jan. 25, 1938. treated and the results subjected to analysis. This has shown that the irradiated areas produced a significantly greater number of spots or cell clusters than appeared on non-radiated areas. It was also found that the new spots appeared chiefly from three to six weeks after treatment with the maximum number arising during the fifth week (see charts, Figs. 6 and 7). In 24 cases two or more cell clusters appeared within the radiated area, in 17 cases only one new spot and EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 195 none were recorded in 11 cases. These results have been compared with the total number of cell clusters appearing on both sides of the body (exclusive of head and fins). The results appear significant even 14. WEEKS AFTER IRRADIATION FIG. 6. Graph of numbers of spots X10 appearing in successive weeks after irradiation. Dotted line, irradiated area. Solid line, other parts of body (head and fins not included). 4 WEEKS AFTEB IRRADIATION FIG. 7. Graph showing same data as Fig. 6 corrected for relative size of areas. Dotted line, numbers in irradiated area X20. Solid line, numbers on other parts of body X-l. 196 H. B. GOODRICH AND J. P. TRINKAUS when no correction is made for the difference in areas compared. When, however, such a comparison is made it is found that the total non-radiated surface examined was approximately twenty times that of the average irradiated area. A graph, incorporating this correction, is shown (Fig. 7) and indicates a notable excess of development of spots in the irradiated areas. During the observational periods, from treatment until 14 weeks thereafter, there appeared a total number of 62 new spots or cell clusters within the irradiated areas and 32 outside of these areas. If we multiply by the factor 20 (20 X 62 = 1240), it appears that had spots appeared at a similar rate in the non-radiated region there would have been 1240 spots, whereas there were only 32. This proportion of nearly 38 : 1 is then an index of the increased reac- tion of the radiated region. It is not impossible that this is an under- estimate. The areas chosen for treatment were frequently below the lateral line, because this region was more clear of melanophores, and it is possible that the ventral region is one having less inherent capacity for production of melanophores. The new cells recorded in the above experiments were in all respects similar to normal melanophores present elsewhere on the fish. Two sets of subsidiary experiments were carried out which, incidentally, gave further confirmation that these cells were normal melanophores. (1) It was found that the melanophores of the hybrid responded very irregularly to an illuminated white environment. In some cells the pigment became concentrated and in others it remained dispersed. New cells arising in the irradiated areas showed this same variability of reaction. (2) Ten scales bearing new cell clusters were transplanted to other parts of the fish as had previously been done by Goodrich and Nichols (1933) with non-radiated fish. The results were similar. The cells lived and in four cases increased, spreading over adjoining scales. DISCUSSION The observations presented in this and the preceding paper (Good- rich and Anderson, 1939) show that the hybrid or calico shubunkin retains the potentiality to produce irregularly situated spots during a considerable part of the life cycle. The radiation appears to stimulate a precocious development of the spots in the areas treated. The question then arises as to what developmental or other conditions con- trol the appearance of these spots or cell clusters. Goodrich (1927), working on the Japanese fish Oryzias latipes, suggested that the varie- gated pattern could be explained by the ameboid migration of pre- determined melanoblasts of two types that producing the maximum EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 197 amount of melanin and the other such a small amount that they re- mained virtually colorless. Recent investigations such as those of DuShane (1935) and Twitty (1936) on amphibia have tended to con- firm the hypothesis of an early determination of wandering chromato- blasts. The paper by Willier and Rawles (1938) on the chick opens the possibility of cell determination and migration in forms where hormones have been shown to be largely operative in other phases of pigment control. The observations of Apgar (1935) on Triturus have suggested the concept of a widespread distribution of colorless chromatoblasts. It, therefore, seems not improbable that we may consider the calico shubunkin (especially Type B of the companion paper) to be invisibly spotted during development with colorless chromatoblasts singly or in nests and that these multiply and differentiate independently at irregular intervals to form the spots or clusters of melanophores. In some respects this hypothesis resembles the old theory of embryonic cell rests advanced to explain the cause of cancer. In certain individual fish a wave of destruction takes place, possibly due to some hormone action, which destroys all melanophores and possibly all melanoblasts in the affected areas. We have never ob- served the appearance of new spots in a region which has suffered such wholesale destruction. Attention should be called to the production in goldfish of pigment cells by X-rays (Smith, 1932). The cells appeared within a few days after treatment and disappeared a few weeks later. They did not seem to be homologous to the pattern-producing cells and resembled cells that had previously been observed arising after various mechanical injuries to the tissues (Smith, 1931). In our own experiments we have noted three cases of the formation of such cells. They were seen on the normal goldfish TT after unusually severe radiation from the mercury arc lamp and the appearance and history of these cells were similar to those noted by Smith. The contrasting reactions of the three genotypes indicate that the hybrid or calico fish retains in adult condition a far greater potency to produce melanophores than either parental form. Goodrich and Han- sen (1931) have pointed out that all three types form melanophores in early development. The ordinary goldfish loses these by wholesale destruction usually at about three months of age, while in the trans- parent shubunkin relatively few ever appear. Neither of these two parental forms produced typical melanophores when irradiated and it may be suggested that melanoblasts have also been destroyed or are 198 H. B. GOODRICH AND J. P. TRINKAUS largely absent. In contrast, the heterozygous type retains the melanoblasts. No attempt is made in this paper to determine what wave-lengths have produced the observed effect. The mercury vapor arc produces a wide range of wave-lengths. The extensive literature on effects of ultraviolet light shows that both stimulating and destructive effects have been observed. Sperti, Loofbourow, and Dwyer (1937), working on yeast cells, have suggested that cells when injured by ultraviolet liberate some growth-promoting substance, thus indicating a possible interrelation of injurious and stimulating effects. The treatments used in our experiments have been relatively more severe than those which have produced primarily stimulating effects on isolated cells. Ultraviolet light penetrates but a few millimeters through animal tissues. Sato (1933) has shown that the ultraviolet light bands charac- teristic of the mercury arc will pass through fish scales. The effect produced in our experiments may well be due chiefly to the regenerative processes following the inflammation and destruction of tissue. SUMMARY 1. Radiation from a mercury vapor lamp produced differing reac- tions in three Mendelian phenotypes. Two parental forms, the ordinary goldfish and the transparent shubunkin, do not develop melanophores as a result of the treatment. The FI hybrid, or calico shubunkin, does respond by an acceleration in the production of new spots or clusters of melanophores. 2. It is suggested that the hybrid during development becomes supplied with colorless chromatoblasts throughout the dermis which are stimulated to precocious multiplication and differentiation as a result of the radiation. BIBLIOGRAPHY APGAR, B. D., 1935. A study of the reappearance of melanophores and the formation of melanophore aggregations (spots) in regenerated ventral skin of the common newt, Triturus viridescens. Jour. Morph., 58: 439-461. DuSHANE, G. P., 1935. An experimental study of the origin of pigment cells in Amphibia. Jour. Exper. Zool., 72: 1-32. GOODRICH, H. B., 1927. A study of the development of mendelian characters in Oryzias latipes. Jour. Exper. Zool., 49: 261-287. GOODRICH, H. B., AND P. L. ANDERSON, 1939. Variations of color pattern in hybrids of the goldfish, Carassius auratus. Biol. Bull., 77: 180-187. GOODRICH, H. B., AND I. B. Hansen, 1931. The postembryonic development of mendelian characters in the goldfish, Carassius auratus. Jour. Exper. Zool., 59: 337-358. GOODRICH, H. B., AND ROWENA NICHOLS, 1933. Scale transplantation in the gold- fish, Carassius auratus. Biol. Bull., 65: 253-265. SATO, N., 1933. Light passing through the scales of fish. Ada Dermatologica, 22: 45. EFFECT OF RADIATIONS ON MENDELIAN PHENOTYPES 199 SMITH, G. M., 1931. The occurrence of melanophores in certain experimental wounds of the goldfish (Carassius auratus). Biol. Bull., 61: 73-84. SMITH, G. M., 1932. Melanophores induced by X-ray compared with those existing in patterns as seen in Carassius auratus. Biol. Bull., 63: 484-491. SPERTI, G. S., J. R. LOOFBOUROW, AND SR. C. M. DWYER, 1937. Proliferation- promoting factors from ultra-violet injured cells. Stud. Instil. Divi Thomae, 1: 163-191. TWITTY, VICTOR C., 1936. Correlated genetic and embryological experiments on Triturus. I and II. Jour. Exper. Zool., 74: 239-302. WILLIER, B. H., AND MARY E. RAWLES, 1938. Feather characterization as studied in host-graft combinations between chick embryos of different breeds. Proc. Nat. Acad. Sci., 24: 446-452. THE REACTIONS OF THE PLANKTONIC COPEPOD, CENTROPAGES TYPICUS, TO LIGHT AND GRAVITY 1 W. H. JOHNSON AND J. E. G. RAYMONT (From the Department of Physiology, McGi.ll University, the Biological Laboratories, Harvard University, and the Woods Hole Oceanographic Institution) INTRODUCTION Field investigations on the vertical distribution of the plankton carried out by many different workers in recent years have established the occurrence of a diurnal vertical migration for most species of the zooplankton. Since most investigators agree in considering light as an important controlling factor, it seemed desirable, following the work of Esterly, Spooner, Clarke and others, to attempt to study the light responses of a single planktonic species under controlled labora- tory conditions. Centropages typicus is a neritic copepod, extremely abundant off Woods Hole at certain times of the year. Clarke (1933) states that the adults show a diurnal vertical migration correlated with changes in the submarine illumination. A few preliminary observations in the laboratory showed us that the adult females are very definitely affected by light. It was therefore decided to conduct experiments on the phototropic and geotropic responses of these animals. Our choice was fortunate in that it was possible to obtain the copepods quickly and easily off Woods Hole, and to keep them at a conveniently low temperature in the laboratory to ensure their healthy existence for at least a few days. PHOTOTROPISM : EXPERIMENTS WITH TUBES HORIZONTAL In order to separate the phototropic from possible geotropic re- sponses, it seemed advisable to test first the reactions of the copepods to light in a horizontal direction. Methods The experimental animals were obtained in Vineyard Sound by towing a scrim plankton net horizontally near the surface for about fifteen minutes. The animals, collected in the glass jar attached to 1 Contribution No. 207 from the Woods Hole Oceanographic Institution. 200 REACTIONS OF COPE POD TO LIGHT AND GRAVITY 201 the net, were poured into 3 liters of sea water and transported im- mediately to the laboratory. The adult female Centropages typicus were selected in diffuse daylight using a wide-mouthed pipette and a binocular microscope. Usually 20 healthy appearing copepods were placed in each of two glass tubes (13 X 2|"), each of which was sealed at one end with a glass plate. The open ends of the tubes were then sealed with similar glass plates, and the tubes arranged in constant temperature tanks maintained at 12 C. in the darkroom. The two experimental tubes could be separated from each other by a distance of 21 feet. It was thus possible to obtain a wide range of light intensities for any one source, by varying the distance between the light source and the tubes. The intensities of the various inside frosted bulbs employed were as follows : 2 Wattage Approximate Intensity at 1 foot 15 13.5 foot-candles 25 25.0 40 43.0 60 75.0 100 150.0 It should be borne in mind that all the light intensities mentioned in the text are only approximate figures. The lowest intensities used were obtained by means of neutral filters in the form of opal discs and white paper, the percentage absorptions of which were obtained by means of a photoelectric cell. Since several filters were used together at the very lowest intensities, corrections were made for diffusion and back-scattering. Each experimental tube was marked off into quarter-lengths, and the distribution of the animals at any time, under any one condition of light, was expressed as the numbers in each section. At the very low light intensities, counting of the copepods was facilitated by lighting the tubes from behind for a moment with a weak red lamp. Preliminary tests made with this lamp showed that it had no effect on the distribution of the animals. 3 In all the experiments, unless otherwise noted, the distribution of the animals was observed at the end of each time interval shown in the tables. After each observation, the tubes were changed, end for end, by turning them slowly in a horizontal plane. This procedure forced the animals to orientate afresh, and to redistribute themselves accord - 2 On the advice of Mr. Eddie Kline, electrical engineer of the Canadian Laco Lamp Co., these can be considered as accurate only within 20 per cent, due to voltage fluctuation. 3 Dr. Horton of the Department of Physics, McGill University, kindly made a spectroscopic photograph of the light emitted and found that the transmission begins o o at 6402 A, and continues beyond 8600 A. 202 W. H. JOHNSON AND J. E. G. RAYMONT ing to the tropistic responses actually in operation during that time interval. Enough time was allowed for the animals to establish their TABLE I Experiment commenced at 4:00 P.M., August 19. Tubes A and B set at distance of 5 ft. and 1 ft. respectively, from source. At 12:00 noon, August 20, tube A moved to 10 ft. At 4:00 P.M., August 20, tube A moved to 20 ft. Tube B was kept at 1 ft. throughout. Source: 60-\vatt lamp. Time Distance Intensity (Positive) * I II (Negative) III IV Aug. 19 4:10 P.M. 5ft. 3.0 f.c. 20 - - - 1 ft. 75.0 f.c. 20 4:40 P.M. 5 ft. 20 1 ft. 20 - - - 6:45 P.M. 5 ft. 16 and 4 _ 1 ft. 20 - - - 9:15 P.M. 5 ft. 10 and 6 2 3 1 ft. 10 and 3 3 - 1 9:30 P.M. 5ft. 8 and 6 2 2 1 ft. 3 and 7 3 3 2 Aug. 20 11:30 P.M. 5 ft. 10 and 3 1 4 3 1 ft. 4 and 8 3 1 1 2:30 P.M. 10ft. 0.75 f.c. 8 and 3 3 3 1 ft. 75.0 f.c. 5 and 9 3 1 1 4:00 P.M. 10ft. 8 and 4 4 4 _. _ 1 ft. 3 and 7 4 1 1 4:45 P.M. 20ft. 0.19 f.c. 9 and 6 _ 3 _ 1 ft. 75.0 f.c. 5 and 10 3 2 1 8:00 P.M. 20ft. 6 and 9 2 2 1 ft. 3 and 9 4 1 2 *Two numbers are sometimes given under Section I (e.g. 16 and 4). This distinguishes those copepods right at the positive end (16), from those still in this section but apparently less strongly attracted. new distribution before a second record was taken, so that their final position was unaffected by the configuration of the previous time interval. Observations A series of tests (Table I) was first carried out in order to determine : REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 203 (1) The normal responses of the copepods to various light intensities within limits found in nature. (2) The effect of continued exposure to constant light intensities over the range studied. The results obtained (Table I) showed that the copepods were positive to all illuminations, and remained largely so after exposure. A number of experiments was then carried out to determine the range of light intensities to which the copepods were sensitive, and to investigate the possibility of the existence of critical light intensities at which the phototropic sign might become reversed. The copepods were found to be positive to low light intensities, the lowest to which they were attracted being ca. 0.005 f.c. (Table II). TABLE II Responses to low light intensities. Distance of experimental tube from source: 20 ft. throughout.* Intensity Time (Positive) I II in (Negative) IV 0.06 f.c. Aug. 20 9:30 P M. 5 and 4 2 3 < i Aug. 21 8:30 A .M. 4 and 6 1 2 1 0.015 10:45 A .M. and 12 - 3 1 ( ( 12:00 Noon 2 and 12 1 2 3 t ( 3:30 P .M. 4 and 5 1 4 0.008 Aug. 24 12:40 P .M. 14 1 3 2 (New animals) 1 ( 2:00 I' .M. 1 and 10 2 2 3 i 1 3:00 P .M. 3 and 9 1 3 3 0.006 3:30 P .M. 11 2 1 3 t t 4:50 P .M. 6 5 - 7 t i 7:00 P .M. 11 2 2 1 I 1 8:00 P .M. 10 - 2 7 u 10:50 P .M. 13 - 4 1 i t Aug. 25 8:30 A .M. 15 1 - - 1 ( 9:30 A .M. 14 - 2 1 0.005 Aug. 26 12:30 P .M. 9 5 4 1 t t 1:55 P .M. 10 4 3 - t ( 2:25 P .M. 3 7 6 3 0.003 Aug. 28 2:30 P .M. 3 4 7 6 (New animals) 1 1 4:45 P .M. 9 4 4 3 1 1 Aug. 29 9:40 A .M. 11 6 2 - 1 1 11:50 A .M. 4 6 3 6 i 1 6:45 P .M. 8 3 3 3 * Each time the light intensity was changed, it was done immediately following the preceding observation. On continued exposure to the much higher light intensities of 150 and 600 f.c. (Table III), the majority of animals on the whole exhibited a positive phototropism, although at times there were more animals in the darker half of the tube and some of the animals apparently became negative on prolonged exposure. 204 W. H. JOHNSON AND J. E. G. RAYMONT It seemed desirable to determine whether the copepods would be repelled by the still higher light intensity (11,380 f.c.) approximating to that of bright sunlight. As a check on the results, other copepods which had been collected at the same time were subjected to a much lower intensity of 4 f.c. The results (Table IV) show that, at least after a short exposure to this very high intensity, half of the animals became negatively phototropic, while the others remained positive. TABLE III Responses to high light intensities Source: 100-watt lamp. Intensity at i ft. : 600 f.c. Intensity at 1 ft. : 150 f.c. Time Distance (Positive) II ill (Negative) IV Aug. 30 5:00 P.M. 1ft. 15 - - 3 1ft. 14 2 3 5:30 P.M. ift. 13 1 1 3 1 ft. 13 - 1 6 6:45 P.M. I ft. 14 1 2 1 1 ft. 10 1 - 8 9:00 P.M. I ft. 14 - 1 2 1 ft. 14 1 2 3 10:15 P.M. ift. 14 2 2 - 1 ft. 10 - 1 8 Aug. 31 9:10 A.M. ift. 5 1 2 10 1ft. 12 3 3 2 10:20 A.M. ift. 8 1 3 7 1ft. 10 - 2 8 11:15 A.M. ift. 11 2 1 4 1 ft. 9 - 2 8 12:15 P.M. ift. 9 1 1 8 1 ft. 10 2 3 6 1:20 P.M. ift. 12 2 - 4 1ft. 12 2 2 4 4:00 P.M. ift. 12 2 - 4 1ft. 8 2 2 8 5:00 P.M. ift. 12 1 3 3 1 ft. 11 2 7 It was rarely that all the animals displayed an invariable reaction (either positive or negative) to any one condition of light. It was possible then that some of the animals were negatively phototropic even though the majority were positive; or again, perhaps some were indifferent. To gain evidence on these points, observations were made on individuals, one being sealed within a tube. At first, observations were made for the most part once every hour, using three widely separated intensities: 3.0, 150, and 600 f.c. REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 205 At the lowest intensity (3.0 f.c.), an individual remained photo- positive for four hours, but appeared to become indifferent after exposure overnight. A second individual was indifferent from the first, and remained so for 15 hours. This behaviour was not modified if the individual was left in darkness, and then exposed to the light. TABLE IV Source: 1000-watt lamp. Tube at 4J inches from source. Control tube at 20 feet. Ice added to aquarium to offset intense heat from source. Time Intensity (Positive) I II ill (Negative) IV 11:40 A.M. 11,380 f.c. 10 2 6 4 f.c. 8 4 1 6 11:55 A.M. 11, 380 f.c. 12 6 4 f.c. 11 2 5 12:05 P.M. 11, 380 f.c. 8 1 - 9 12:20 P.M. 11, 380 f.c. 10 _ 4 6 4 f.c. 13 2 3 1:20 P.M. 11,380 f.c. 8 1 2 10 4 f.c. 11 2 2 3 1:35 P.M. 11,380 f.c. 9 1 3 8 1:45 P.M. 11,380 f.c. 8 1 3 8 4 f.c. 15 2 2 - 2:00 P.M. 11,380 f.c. 9 - - 9 2:10 P.M. 11,380 f.c. 10 1 1 7 4 f.c. 14 2 1 1 2:25 P.M. 11, 380 f.c. 11 1 - 7 2:40 P.M. 11, 380 f.c. 8 1 11 4 f.c. 14 2 1 1 The responses of two individuals at an intensity of 150 f.c., and of two others at 600 f.c. were such that one individual at each intensity remained positive for 24 hours, while the other individuals were posi- tive for the first 5 hours but apparently became indifferent after exposure overnight. More extensive experiments on individuals were carried out, mak- ing observations every ten minutes, so long as it was possible to do so, 206 W. H. JOHNSON AND J. E. G RAYMONT over a long period of time, and at a wide range of intensities (600, 150, 75, 67, 33, 13.5, 2.4, 0.87, 0.03, 0.006, and 0.002 f.c.). Of four individuals (A, B, X, and F), specimens B and F were strongly and constantly photopositive to all the above intensities; indeed, specimen B was never recorded outside Section I. Individual A was in the main attracted although less so at intensities above 75 f.c. Individual X, although less consistent, was generally attracted by the light, but occasionally at both high and low intensities it was found at the negative end of the tube, even from the beginning of the experiment. Having studied the effects of continued exposure to different intensities, it was decided to determine the effect of changing light intensity a condition which is more like that which occurs in nature. The changes in intensity were obtained by varying the position of the source relative to the two experimental tubes. Thus the quality of the light remained unchanged, and two experiments could be carried on at once. Successive experiments were carried out by moving the source first 1 foot, then 2, 5, 10, and finally 20 feet every ten minutes (owing to difficulties in counting, 15-minute intervals were sometimes unavoid- able). The intensities ranged from 11,380 to 4 f.c. Before the experiments were commenced, the tube at the maximum intensity was left exposed to light until a considerable percentage of the animals exhibited repulsion. Regarding the one-foot changes: On increasing the intensity from 4 to 11,380 f.c., the animals remained continually attracted showing always at least 80 per cent in the positive half of the tube. However, after continued exposure for one hour at the highest intensity, only 40 per cent were still attracted. In the opposite tube, 55 per cent of the copepods were repelled at the beginning when the intensity was 11,380 f.c., and it was necessary to decrease the intensity to 64 f.c. before 80 per cent of the animals were attracted. Considering the results of the 2 ft. changes, it was found that essentially similar conclusions could be reached. In the increasing intensity experiment, actually 100 per cent of the animals exhibited constant positive phototropism. Decreasing the intensity resulted in progressive attraction down to 16 f.c., when about 80 per cent of the animals were in Sections I and II. Further decreases caused little change. The 5, 10 and 20-foot changes may be considered together. Re- garding the increasing intensities, it is striking that none of the changes had any effect on altering the original distribution of the animals. The numbers of animals in each half of each tube remained almost perfectly REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 207 constant with the ten-minute intervals allowed, and it was only after prolonged exposure (45 to 60 minutes) at 11,380 f.c. that repulsion was brought about. Of the decreasing intensity experiments, in the 5-ft. changes progressive attraction resulted in 80 per cent of the animals being positive at an intensity of 7 f.c. Progressive attraction also resulted in the other experiments, with 70 per cent of the animals being attracted in the 10-ft. changes at the minimum intensity of 4 f.c. (After one hour at 4 f.c., 80 per cent were positive.) All these experiments on different magnitudes of decrease, each occurring with 10-minute intervals, would seem to indicate that the greater the magnitude of change, the lower the intensity at which a large number of the copepods became positively phototropic. This statement may be misinterpreted unless it be remembered that un- doubtedly 80 per cent, or more, of the copepods would have migrated to the positive half of the tube at much higher intensities had more time been allowed before the next change was made. (There would thus appear to be a "time-lag" effect.) The above experiments show the effects of different magnitudes of increase and decrease with a constant time interval of 10 minutes. The percentage relationship between any one intensity and that which immediately preceded it is not by any means constant during any one succession of changes. Thus experiments were next conducted similar to the foregoing, except that there was a constant percentage increase or decrease throughout each series of changes. The actual rates of change used were such as may occur in nature. (The values chosen were the maximal changes observed by Clarke (1933) at one station in the Gulf of Maine.) Increases and decreases of 10 per cent per hour were first tried, through a range of high intensities (11,380 to 2,840 f.c.), and then through a low intensity range (9.5 to 6.2 f.c.). Considering first the decreasing intensities, through the high range there was progressive attraction, while through the low range there was practically no alteration in the distribution. As regards the increasing intensities experiment, there was little observable change, but, if anything, a rather larger percentage of animals was attracted with time. The same result was obtained with the low intensity range. Decreases and increases of 20 per cent per hour, through both high and low intensity ranges, gave similar results. PHOTOTROPISM AND GEOTROPISM: EXPERIMENTS WITH TUBES VERTICAL Parker, Dice, Esterly, Clarke and others have demonstrated that geotropism is frequently an important factor in the vertical migration 208 W. H. JOHNSON AND J. E. G. RAYMONT of plankton. It seemed desirable, therefore, to carry out experiments using vertical tubes to ascertain whether the light responses would be different, and to test for the occurrence of a true geotropic reaction. Methods The aquaria were replaced by two large bell-jars held upright by specially constructed wooden stands. The same experimental tubes were used, but they stood vertically in all the following experiments. A lamp was suspended over each tube, and, by means of a pulley system, the distance between the lamp and the tube could be quickly altered. The maximum distance thus obtainable was 4| feet. When- ever it was desired to illuminate the animals from below, the tubes were simply placed upright on an iron tripod, and the lamp placed underneath. Observations It was decided to find the effects of various rates of change of light intensity, and to compare the results with those obtained in the horizontal experiments. Unfortunately the 1,000-watt lamp burned out and as it was impossible to replace it in the short time remaining, it was necessary to confine the indoor experiments to the lower light intensities (0.67 to 240 f.c.). A wide variety of rates of change was used: 25 per cent per hour, and 25, 50, 100, 300, and 700 per cent per half-hour. Considering the experiments on increasing light intensities the following conclusions were reached. Within the range of intensities used, it seemed that, in general, increasing the light at a variety of rates does not bring about repulsion. One experiment, however, using 25 per cent increases per half-hour, through a range from 7.4 to 19.1 f.c. did cause repulsion: 70 per cent of the copepods were attracted initially, but as the intensity increased, fewer remained positive until only 16 per cent were attracted at 19.1 f.c. A large number of other experiments, however, at intensities near 7.4 to 19.1 f.c. (also at higher and lower ranges, and at rates from 10 per cent to several hundred per cent) was carried out, and in no other case was this repulsion observed. In the great majority of cases the distribution remained almost constant. It may be then, that this single case of repulsion does not demonstrate the normal behaviour of these animals, at least under laboratory conditions. In the experiments on decreasing light intensities, with the excep- tion of a single experiment, decrease in intensity at all rates, and through all the ranges of intensity employed, resulted in more and more of the animals swimming to the top of the tube as the light diminished. REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 209 This progressive attraction was sometimes very great. For example, in two experiments only 25 per cent of the copepods were positive at the beginning, and nearly 90 per cent at the end. The exceptional experiment was the only one employing so low a rate of decrease as 10 per cent. It is possible that such changes are too slow to be per- ceptible to the animals (below threshold). It was thought desirable to determine the effect of increasing light intensity, using direct sunlight, so that a very high intensity range would be available. The experiment was conducted in the open behind the Oceanographic Institution. An inverted bell-jar was used as in the darkroom, with the experimental tube placed inside it, standing vertically. Since it was here impossible to circulate cooled water through the bell-jar, it was simply refilled with cold sea water whenever the temperature rose. The stand holding the bell-jar was completely TABLE V Reactions to direct sunlight Time No. of Opals Relative Sunlight Approximate Int. in Tube (f.c.) (Top) II III (Bottom) IV per cent 12 Noon 4 100 1,080 12:30 P.M. 4 100 1,080 11 1 4 3 1:00 P.M. 3 98 1,400 11 1 3 3 1:30 P.M. 2 93.6 2,000 12 1 1 4 2:00 P.M. 1 88 2,640 19 - - 1 2:30 P.M. 79 9,470 11 2 1 5 3:00 P.M. 73 8,760 11 ' - 1 5 3:30 P.M. 4 59 636 12 - 3 3 4:00 P.M. 2 43.6 935 5 2 6 4 4:45 P.M. 27 3,240 7 1 3 7 covered with black tar-paper. A small aperture cut in the top allowed a beam of sunlight to fall on the top of the experimental tube. On one side of the stand, the tar-paper formed a moveable flap which could be lifted, and the necessary counts made. Four opal diffusing discs were placed over the aperture to reduce the light; these were removed at intervals. In the first experiment, they were removed one at a time, in the second two at a time, and in the last experiment all four were removed together. Each disc alone transmits 25 per cent of the light falling upon it. The beam of sunlight was directed on to the aperture above the tube by means of a simple plane mirror which could be turned as the sun changed its elevation. The light intensity was measured by means of a Weston Photronic Cell. The results of the experiment (Table V) show that when the light had increased from about 1,000 to about 9,000 f.c. over a period of two 210 W. H. JOHNSON AND J. E. G. RAYMONT hours, the animals were at all times strongly photopositive. How- ever, increases starting at lower intensities resulted in a majority of the animals in the lower half of each tube. Is there also a negative geo- tropism which becomes stronger with increase in light intensity? Certainly the results indicate that mere rate and direction of change of light alone cannot account completely for the movements of Centropages typicus. Thus experiments were next carried out in order to test the possi- bility that the copepods might react to gravity, and that the above results were only partially due to phototropic responses. The experimental tubes were placed vertically in the bell-jars in the normal way. The animals were then left in darkness, and counts made later with the red lamp. For example, the tubes were left for 1^ hours in darkness and subsequent counts gave the following results (Tubes A and B were treated identically to furnish checks on each other) : (Top) (Bottom) I II III IV Tube A 14 4 1 Tube B 64 8 The tubes were then reversed vertically end for end. After one- half hour the following results were obtained : (Top) (Bottom) I II III IV Tube A 12 5 3 TubeS 12 3 3 The tubes were again reversed. After one-half hour the following results were obtained : (Top) (Bottom) I II III IV Tube A 11 3 1 2 Tube B 15 1 3 The above results clearly show that the animals are on the whole negatively geotropic in darkness. Careful observation showed that the animals sink rapidly if they cease swimming. Hence actual effort was necessary for them to remain at the tops of the tubes, and the geotropism must then be quite strong. The relation between geotropism and phototropism was then tested by taking the above animals from darkness and illuminating them from below, with the following results: (Top) (Bottom) I II III IV Tube A (15-watt lamp below) 7 4 3 4 Tube B (100-watt lamp below) 1 - 19 REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 211 The results would indicate that negative geotropism is stronger than positive phototropism when the light is weak, while positive phototropism is overwhelmingly strong when the intensity is high. Tube B was returned to darkness and a count 15 minutes later showed that the majority of animals were in Section I. These results verified the negative geotropism. A 60-watt lamp was then placed below the tubes and the following results obtained : (Top) (Bottom) I II III IV Tube B (60-watt lamp below the tube) 1 19 Both tubes were again returned to darkness and a count 45 minutes later again showed a large majority exhibiting negative geotropism. A 25-watt lamp was then placed below the tubes: (Top) (Bottom) I II III IV Tube B (25-watt lamp below the tube) 4 1 3 9 The experiment was repeated. The animals again showed negative geotropism in darkness. With a 25-watt lamp below the tubes the results were as follows: (Top) (Bottom) I II III IV Tube B (25-watt lamp below the tube) 4 1 12 Finally it was decided to determine the effect of replacing a low light intensity by a high one, when the geotropism and phototropism were in opposition. It has been shown that after exhibiting negative geotropism in darkness, on exposure to a 25-watt bulb from below the distribution was: (Top) (Bottom) I II III IV Tube B (25-watt lamp below) 4 1 12 This lamp was then replaced by a 100-watt lamp. A count after 15 minutes showed : (Top) (Bottom) I II III IV Tube B (100-watt lamp below) - 17 All the above experiments definitely establish that the adult female Centropages is primarily negatively geotropic and positively photo- tropic. When the two are acting in opposition, the positive photo- tropism becomes progressively stronger as the light intensity increases. DISCUSSION It is still a controversial matter how far laboratory experiments of the type conducted are applicable to conditions in nature. Through- out all the experiments, however, it was our aim to avoid "shock" 212 W. H. JOHNSON AND J. E. G. RAYMONT conditions, and the use of surface tow-nettings avoided large changes in light intensity during the collections. It would seem from the experiments with artificial light, that adult female Centropages typicus should be right at the surface during most of the day, since they are strongly positively phototropic to a very wide range of light intensities, and it does not seem that continual decreases are always necessary to cause a majority to remain positive, such as was found to be the case with Acartia clausi (Johnson, 1938). However, repulsion does occur to some extent on prolonged exposure to very high intensities, and also in the experiments using direct sun- light (Table V) when the illumination increased through such ranges of low intensities as may occur in the early morning. Hence, after considerable exposure to strong sunlight (about midday in summer) and possibly also when the light is increasing in the early morning, Centropages might be expected to be a little lower in the water. G. L. Clarke (1933) however, found that these copepods have a maximum of about 13 m. during most of the day in the Gulf of Maine. Some hauls made in August, 1935, near Woods Hole, were examined and these in general confirmed this finding, although there were cases when the majority were at the surface. (Clarke also did find, for two stations, the majority at the surface.) In considering this difference it must be remembered that there are other factors acting in nature. Thus, especially at the surface, turbulence may carry the copepods to somewhat lower depths. Further, the possibility of muscular fatigue must not be overlooked. As has been mentioned, Centropages will sink rapidly as soon as it ceases swimming, and thus some will tend to sink below the surface, though positively phototropic. This probably accounts for the observation, that, although using the same intensities, a considerably larger percentage of animals is found in the negative half of the tube in the vertical experiments than in the horizontal ones. It should also be noticed that Clarke did find a secondary maximum of Centropages at the surface. The rise to the surface at night, observed by Clarke and others, is explainable since Centropages is always very strongly attracted when the light intensity is diminished. The negative geotropism, evident at least during and just after exposure to darkness, will aid the rise. Parker (1901) found that female Labidocera migrate surfacewards at night due to positive phototropism and negative geotropism, and Dice (1914) considered geotropism the major factor in the migration of Daphnia. However, the recent findings of Kikuchi (1938) exemplify REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 213 the fact that the actual r61e played by geotropism probably varies from species to species. Since Centropages is positively phototropic to very low intensities, the upward migration will presumably continue when the light is exceedingly weak. Further, when the animals have reached the surface, they will tend to remain there during darkness owing to the negative geotropism, and they will not take up a more or less uniform distribution, as Russell has supposed for some planktonic species. As regards the downward migration in the morning, we were gen- erally unable to demonstrate repulsion with increase in light intensity using electric light in the laboratory. However, in the experiments using direct sunlight, it was shown that increase in intensity at a low intensity range from about 700 to 3,500 f.c., did cause repulsion, and this range of light change might be expected in the early morning. It is possible that exposure to darkness during the night might also tend to render the animals more sensitive to light, but there is the opposing geotropism to consider. This has been shown, however, to be definitely weaker for average light intensities. Further experi- ments, however, are desirable in this connection. Although no experiments were conducted to test specifically Ester- ly's theory of a diurnal rhythm (Esterly, 1917, 1919), it would seem from an examination of our readings at different times of the day that such a rhythm is absent in Centropages. Rose considers that a species exhibiting diurnal vertical migration is adapted to a certain optimum light intensity (Rose, 1925). Many investigators have been unable to demonstrate such optima in the laboratory. Esterly, for example, found Calanus negative to all intensities used, provided the tempera- ture was above 10 C. Rose believed that if a wide range of intensities was employed in the experiments, the optima would be demonstrable. We therefore used a very wide range in our experiments, but did not find any such optimum for Centropages. Reversal of phototropic signs with absolute intensity of light was also difficult to obtain, though Loeb, Parker, Rose, etc. have demon- strated this for many planktonic species. It should be noted that Clarke also found there was no evidence from his experiments for an optimum light intensity in Daphnia. He also found that reversal of phototropic sign could not be brought about by absolute light intensity in this form. (Clarke, 1930 and 1932.) Various authors have frequently pointed out the complexity of the problem of vertical migration by showing differences in behaviour between different species (e.g. Clarke, 1933), between the sexes of a single species (e.g. Russell, 1928), and between ages of the same sex 214 W. H. JOHNSON AND J. E. G. RAYMONT of one species (e.g. Gardiner, 1933). The observations of the authors of the present paper further illustrate that although the majority of adult female Centropages typicus do behave in a similar manner, varia- tion in vertical distribution between individuals may be expected even when they are of the same species, sex and age. This is in agree- ment with field studies. SUMMARY Experiments on phototropism and geotropism in adult female Centropages typicus were conducted. The following conclusions were indicated : A. Experiments with experimental tube horizontal. 1. The copepods are primarily photopositive and constant exposure does not modify this reaction except at very high intensities in the neighborhood of that of bright sunlight (ca. 12,000 foot-candles) when a large number exhibited negative phototropism after continual exposure for about an hour. 2. The lowest intensity at which there were always more copepods in the brighter than the darker half of the tube was ca. 0.005 f.c. 3. There are two types of individuals. One type, after continuous exposure to light, becomes indifferent. In the other type, the animals are persistently attracted. 4. Decrease in light intensity, at a variety of rates and at a wide range of intensities, always results in increased attraction. 5. Increase in light intensity, at a variety of rates and at a wide range of intensities, has no effect on the behaviour. Only prolonged exposure at high intensities repels the animals. B. Experiments with experimental tube vertical. 1. With the light from above the animals stay mainly at the top of the tube through a wide range of intensities, a distribution which is probably the result of positive phototropism, negative geotropism, or both. 2. Increases in intensity have no effect on the animals except when sunlight is used. A fair percentage of the animals is then repelled. 3. With the exception of decreases as low as 10 per cent per hour, decreases in intensity result in increased attraction. 4. The animals are strongly negatively geotropic in darkness. When geotropism and phototropism are opposed, the reactions depend upon the intensity of the light. 5. The possible bearing of these conclusions on the vertical distribu- tion and diurnal vertical migration of adult female Centropages typicus is discussed. REACTIONS OF COPEPOD TO LIGHT AND GRAVITY 215 BIBLIOGRAPHY CLARKE, G. L., 1930. Change of phototropic and geotropic signs in Daphnia induced by changes of light intensity. Jour. Exper. Biol., 7: 109-131. , . - ., 1932. Quantitative aspects of the change of phototropic sign in Daphnia. Jour. Exper. Biol., 9: 180-211. CLARKE, G. L., 1933. Diurnal migrations of plankton in the Gulf of Maine and its correlation with changes in submarine irradiation. Biol. Bull., 65: 402- 436. DICE, L. R., 1914. The factors determining the vertical movements of Daphnia. Jour. Animal Behavior, 4: 229-265. ESTERLY, C. O., 1917. The occurrence of a rhythm in the geotropism of two species of plankton copepods when certain recurring external conditions are absent. Univ. Calif. Publ. Zool., 16: 393-400. , . ., 1919. Reactions of various plankton animals with reference to their diurnal migrations. Univ. Calif. Publ. Zool., 19: 1-83. GARDINER, A. C., 1932. Vertical distribution in Calanus finmarchicus. Jour. Mar. Biol. Ass'n., N.S., 18: 575-628. JOHNSON, W. H., 1938. The effect of light on the vertical movements of Acartia clausi (Giesbrecht). Biol. Bull., 75: 106-118. KIKUCHI, K., 1938. Studies on the vertical distribution of the plankton Crustacea. Records of Oceanogr. Works in Japan, 10: 17-41. LOEB, J., 1908. Uber Heliotropismus und die periodischen Tiefenbewegungen pelagischer Tiere. Biol. Zentral., 28: 732-736. PARKER, G. H., 1901. The reactions of copepods to various stimuli and the bearing of this on daily depth-migrations. Bull. U.S. Bur. Fish., 21: 103-123. RUSSELL, F. S., 1928. The vertical distribution of marine macroplankton. VII. Observations on the behavior of Calanus finmarchicus. Jour. Mar. Biol. Ass'n., N.S., 15:429-454. ROSE, M., 1925. Contribution a 1'etude de la biologie du plankton. Le probleme des migrations verticales journalieres. Arch, de Zool. exper. et gen., 64: 387-542. EMBRYONIC INDUCTION IN THE ASCIDIA S. MERYL ROSE (From the Department of Zoology, Columbia University, and the Marine Biological Laboratory, Woods Hole, Massachusetts) INTRODUCTION The Ascidia are grouped with those animals whose early develop- ment is termed mosaic. Yet, in the closely related Vertebrata, organs form as the result of interaction between those cells which become the definitive organ in question and neighboring cells, whose descendants take no part in the actual formation of the organ.. The independent differentiation of organs in the Ascidia and the dependent differentia- tion of the same organs in the Vertebrata presents a problem. It is believed that the Ascidia and the Vertebrata are descendants of a common ancestor which contained organs similar to those now common to both groups. The nerve cord, for example, in the two groups is thought to be homologous. The common ancestor must have had a nerve cord which arose either under the influence of inductors or independently as a mosaic piece. It seems strange that in the course of evolution the vertebrate nerve cord and the ascidian nerve cord could have remained such similar embryonic structures, when their modes of origin were diverging so greatly that the one now forms under the influence of inductors and the other quite independently of induc- tors. Possibly this difference in mode of development between the two groups is more apparent than real. The injury experiments of Conklin (1905&) established the fact that in Styela partita surviving blastomeres do not deviate from their prospective potency by regulating to form more morphological units than they normally form as parts of a whole embryo. Conklin's work further showed that differences in protoplasmic appearance and cleavage peculiarities develop in the uninjured blastomeres just as they would were the blastomeres part of an intact embryo. These differ- ences are numerous enough to allow a careful observer to differentiate the presumptive tissues and organs before the formation of the defini- tive structures. This fact, I think, is the basis for the belief that the ascidian egg is a mosaic of self-differentiating parts. The isolated parts certainly self-differentiate into what are recognizably distinct presumptive regions, but the question is whether any or all of these isolated presumptive regions are capable of further self-differentiation into embryonic organs. 216 EMBRYONIC INDUCTION IN ASCIDIA 217 The problem, then, is to determine whether or not inductive influ- ences are present in the developing ascidian embryo, and, if they are present, which cells release inductors and which structures develop in dependent fashion. The answers have been sought with the aid of iso- lation and transplantation techniques. I wish to express deep gratitude to Dr. Barth for his valuable sug- gestions during the course of this work. MATERIALS AND METHODS The animal chosen for these experiments was Styela partita. This particular animal was used because its normal embryonic development is comprehensively portrayed and because the mapping of presumptive regions is complete (Conklin, 1905a). A further reason for using the egg of Styela is that much of the experimental work on early ascidian development with which the present work must be compared was done on this egg. Fertilized eggs were obtained in two ways. In the early part of this work several animals were cut in two and the eggs and sperm removed from the gonads and ducts in a pipette and mixed in sea water. Only a small percentage of eggs was fertilized. These were recognized by the cap of concentrated yellow pigment which forms after fertilization and were sorted out for use. This method is laborious and leaves little time before the first cleavage occurs in which to prepare the eggs for operations. Fertilized eggs are obtained more easily and quickly from spawning animals. Usually Styela spawn in the laboratory some time between 4 and 7 P.M. However, they may be induced to spawn at any time of day or night by subjecting them to light for eleven or twelve hours preceding the desired time of spawning. The animals were kept in running sea water aquaria where the light was controlled with an opaque oil-cloth cover and an electric light. Bulbs of 40 and 150 watts placed directly over the tank and about eighteen inches from the animals were found to be equally effective. As a rule the aquarium was shaded with the oil-cloth during the afternoon and evening and the light turned on at about 10 P.M. The animals then started to spawn the next morning between nine and ten. The time between the spawning of the first animal and the last varied from fifteen minutes to several hours. During the longer spawning periods eggs were collected for use several times. The same group of animals could be induced to shed clouds of sperm and eggs on four or five successive days, by con- trolling the illumination. There is something released into the water by the spawning animals 218 S. MERYL ROSE which induces others to spawn, provided that the latter have had almost the necessary eleven or twelve hours of light. This knowledge was used occasionally in causing the animals of one tank to spawn several hours before the expected time by adding some water from another tank in which spawning had ceased shortly before. Eggs were carried in small pipettes through eight washes of pasteur- ized sea water. The water had been heated to 70 C. and maintained at that temperature for five or ten minutes. After cooling it was aerated by shaking and used immediately or kept in the refrigerator overnight until shortly before use the next day. The operating dishes were 20 mm. Stender dishes. These were flamed with a Bunsen burner each time before use and a hot 1.5 per cent agar solution in sea water was permitted to cool and solidify upon the bottom of each dish. The smooth agar surface prevented the eggs from adhering to the glass. The operating solution was 0.4 per cent 0. IN HC1 in pasteurized sea water, which changes the pH to approximately 7.6. The sea water was slightly acidified because most of the eggs, after removal from the membranes in ordinary sea water, cleaved abnormally and often cleavage furrows disappeared although nuclear divisions continued, very much as is the case when Arbacia eggs are treated with alkaline or acid sea water (Smith and Clowes, 1924). Acid rather than base was tried because Child (1927) had found more normal development of Corella willmeriana embryos outside of the atrium when the CC>2 tension was increased. Child found the pH of the atrium to be ap- proximately 7.4. In the acidified sea water injury from manipulation was much less frequent. The eggs seemed more viable and, without membranes, could develop into normal tadpoles not distinguishable from those grown within the protective membranes. Pasteurized sea water and semi-sterile precautions with operating dishes and instru- ments were employed because survival with good differentiation was increased from about 10 per cent to over 90 per cent by so doing. Instruments were dipped in alcohol between operations and the pipette shaft flamed each time after use. Membranes were removed from the eggs in operating dishes with the aid of fine watchmakers' forceps. The denuded eggs were then transferred to other operating dishes in finely tipped pipettes. Blasto- meres were separated with Spemann glass needles. Transplantations were accomplished by simply bringing one cell or group of cells to rest upon another group with which combination was desired. The cells of the cleavage stages are quite sticky and adhere readily. Embryos were fixed in Bouin's fluid. After fixation they were transferred to a 1.2 per cent agar solution as it was cooling. After solidification of the agar, blocks containing the embryos were cut out EMBRYONIC INDUCTION IN ASCIDIA 219 and passed through the alcohols. The 95 per cent alcohol through which the blocks were passed during dehydration contained some water-soluble eosin. The blocks and the embryos were stained enough so that they might be seen more easily during clearing and imbedding. This is a modification of a technique employed by Dalcq (1932) for the manipulation of ascidian embryos. Sections of seven micra were cut and then stained a few minutes in Heidenhain's haematoxylin at 45 C. after a previous mordanting of twenty minutes in 4 per cent ferric alum. Further staining for three minutes in 1 per cent light green after treatment in 0.5 per cent phosphotungstic acid for five minutes was sufficient to stain the yolk material. A 0.5 per cent solu- TABLE I Stage Presumptive Value and Cell Lineage of Cells Used in Experiments 16 43 44.1 45.1 32 "46.1 46.2 45.2 "46.3 46.4 c4.2 "a5.3 a5.4 B3 '54.1 M.2 Presumptive Value Endoderm Notochord and Spinal cord Endoderm and Mesenchyme Notochord and Spinal cord Cerebral vesicle and Epidermis Endoderm and Mesoderm Epidermis tion of eosin in slightly acidified 95 per cent alcohol counterstained sufficiently in thirty seconds. The photomicrographs were taken through an oil immersion lens. I wish to thank Mr. J. Godrich for his part in the preparation of the photographs and plates. EXPERIMENTAL SECTION In Table I the presumptive value and cell lineage of the cells used in the experiments to be described are given. The presumptive value and cell lineage were worked out by Conklin (1905o.). The relative positions of the cells described in Table I may be seen in Figs. 1-4. 220 S. MERYL ROSE In both the table and figures the cell notations are given for only one side of the embryo, since the cleavage pattern is bilaterally symmetrical. When reference is made to corresponding cells of both sides of the embryo, the figure 2 is placed before the cell lineage notation. A figure greater than 2 indicates that corresponding cells of more than one embryo have been used. PLATE I Abbreviations: A, anterior; An animal pole; P, posterior; Veg, vegetal pole. The cell lineage notations may be understood by referring to Table I. FIG. 1. A vegetal view of a four-cell stage. FIG. 2. A right side view of an eight-cell stage. FIG. 3. A vegetal view of the eight vegetal cells of a sixteen-cell stage. FIG. 4. A vegetal view of the sixteen vegetal cells of a thirty-two cell stage. FIGS. 5-8. Surface views of anterior half, 2A3, embryos. Supernumerary pigment spots are present in all and a bare notochord is shown in Fig. 6. FIG. 9. A surface view of an anterior quarter embryo, 1.43. FIG. 10. A surface view of an anterior vegetal quarter embryo, 2^14.1. A notochord is present. The early cleavages allow an experimental isolation of the pre- sumptive regions and combinations of various regions in order that the normal interactions may be ascertained. Comparison of Anterior and Posterior Half Embryos Separation of the yellow, B3, and gray cells, A3, in the four-cell stage serves to test to what extent the two may differentiate inde- EMBRYONIC INDUCTION IN ASCIDIA 221 pendently of each other. Both parts have been shown, by Chabry (1887) in Ascidiella, and by Conklin (19056) in $tyela, to undergo partial cleavage and to gastrulate. The anterior or gray cells may form a notochord, neural structures including the pigmented sensory cells (otolith and eye-spot), and endoderm which in some cases becomes arranged in the form of a gut with lumen. Figure 13 is a drawing of a section of an anterior half embryo showing the above-mentioned features. The high degree of differentiation of the anterior partial embryos is in agreement with the results of Chabry and Conklin. A peculiarity shown by approximately half of these anterior half embryos, grown from 2^13 cells, is the presence of more than the normal two sensory spots. Figures 5-8 show surface sketches of four such embryos. Figure 9 shows an anterior quarter embryo grown from 1^43. The greatest number of pigment spots observed in the anterior half embryos was nine. Often pigment formed in cells widely sepa- rated, sometimes on opposite sides of the embryo. In many of the anterior embryos the neural plate did not fold over to form a cerebral vesicle, but, instead, remained on the surface of the embryo. This was usually the case when supernumerary sensory spots were formed. Figures 15 and 16 are adjoining sections of a 2^43 embryo which has an infolded embryonic nervous system. One of the pigment spots is ex- ternal and three are internal. One of the internal sensory cells was cut in such a way as to be included in both sections. It is readily seen that more pigment is produced by anterior half embryos than would be produced by such cells when part of a whole embryo. The presence of extra sensory cells has also been observed in unoperated embryos. Here, however, their occurrence is rare, and never more than four have been seen in one embryo. The phenomenon of supernumerary pigment spots will be further discussed below. Contrasted with the rather complete differentiation of the anterior half embryo is the unorganized condition of the posterior half. Gastru- lation occurs and the embryos survive past the time when the controls become swimming tadpoles, but the presumptive muscle cells remain large and almost round, never elongating nor taking on the fibrous appearance of muscle cells. Figure 11 is a drawing of a section of a posterior half embryo showing the absence of differentiation. Chabry (1887) cultured posterior half embryos of Ascidiella and found poorer development of posterior than of anterior halves. No mention was made of muscles. Conklin (1905a, p. 52, footnote), employing the convention of calling a cell a "muscle cell" if, in normal development, it would give rise to nothing but muscle, designated these undifferen- tiated cells of the partial embryos, muscle cells. Since this work is 222 S. MERYL ROSE II 12 13 PLATE II Abbreviations: 5, eye-spot; Ep, epidermis; E, endoderm; G, gut; M, mesoderm; N, neural tissue; No, notochord; 0, otolith. FIG. 11. A drawing of a section of a posterior half embryo, 2B3, containing epidermis, mesoderm and endoderm. FIG. 12. A drawing of a section of a posterior vegetal plus anterior animal embryo, 254. 1 + 2a4.2, containing epidermis, mesoderm and endoderm. FIG. 13. A drawing of a section of an anterior half embryo, 2.43, containing epidermis, gut, notochord and neural tissue with otolith and eye-spot. EMBRYONIC INDUCTION IN ASCIDIA concerned with the problem of differentiation, such cells are considered as presumptive, and the term "muscle cell" is reserved for those cells which attain the stage of differentiation found in contractile tissue and acquire myonbrillae. The isolation and study of twenty-seven anterior and posterior embryos have shown that the gray cells, the A3, contain within them- selves the ability to self-differentiate, whereas the yellow cells, the B3, lack something which would enable them to differentiate. Animal and Vegetal Embryos It is possible to observe the development of presumptive epidermal and cerebral vesicle cells isolated from mesodermal and endodermal cells by separating the animal from the vegetal blastomeres in the eight-cell stage. In forty-five such cases there was never evidence of neural differentiation in either the animal or vegetal half. Nothing like a neural tube formed, nor did sensory cells develop. The picture one obtains from sections of partial embryos of the animal region is one of undifferentiated cells showing no cerebral vesicle (Figs. 17-19). Instead of a row of epidermal cells surrounding a vesicle of neural tissue bearing pigment spots in two of the cells, the isolated animal embryos show nothing but a group of closely packed similar cells usually arranged about a cavity. This cavity formed between the dividing cells before the time of formation of neural tissue at the time when control embryos were gastrulae. Some of the animal embryos have a wrinkled appearance and instead of a single cavity, contain several. Tung (1934), performing the same operation in Ascidiella scabm, obtained animal embryos, some of which he believed contained neural tissue. These embryos showed folds or depressions, the cells of which stained more heavily with eosin than did the other cells, or contained a few cells grouped together making a small tube. Since the cerebral vesicle in normal embryos stains more readily with eosin than do the other tissues, and since neural structures arise through a folding proc- ess, Tung thought his animal embryos possessed neural tissue. The stain criterion may be reasonably doubted. Tung shows that the presumptive neural cells in the gastrula stage are already eosinophil. At this time the cells are undifferentiated. It seems inadvisable, therefore, to use the eosinophil nature of the cells as a criterion of neural differentiation. Conklin (19056) also recognized neural tissue in isolated anterior animal cells, but used different criteria. His criteria were that the cells in question in the living condition were very clear cells, as are the 224 S. MERYL ROSE neural plate cells of a whole embryo, and, further, that their cell lineage and size were the same as the neural plate cells of a whole embryo. These criteria of neural differentiation seem valid for only the very beginning of differentiation of neural cells. A better criterion would be the formation of a structure more like the normal cerebral vesicle, a vesicle bearing an otolith or an eye-spot. Such has never been recorded from isolated animal cells. Never in past work (Chabry, Conklin, Tung), nor in the present work have sensory structures been seen to develop in isolated animal cells. It seems, then, that there must be some factor extrinsic to the presumptive brain cells which enables them to differentiate. Isolation of the vegetal quartet of blastomeres, 2^44.1 -f- 254.1, in the eight-cell stage should test whether vegetal cells are able to self- differentiate. Few vegetal half embryos survived until the time when differentiated structures might be expected. The great majority con- tinued to cleave until gastrulation time. Then the embryos became loosely adhering masses of cells which soon disintegrated. One, how- ever, remained intact long enough to produce a differentiated noto- chord. The vegetal embryos of Ascidiella produced by Tung (1934) show a higher degree of differentiation. Notochordal cells have also been seen to form in quarter embryos derived from the anterior vegetal cells alone, the 2^44.1. Figure 9 is a surface view of such an embryo showing the bare notochord. Abbreviations: A, cup of animal cells; M, myofibrillae; No, notochord; Ot, otolith; V, plug of vegetal cells. FIG. 14. A section through the cerebral vesicle of an unoperated tadpole, showing the size of the larger pigment spot, the otolith. FIG. 15. A section through the pigment spot region of an anterior half embryo, 2.43. FIG. 16. An adjoining section of the same embryo shown in Fig. 15. FIG. 17. A section through an animal half embryo, 2a4.2 -+- 264.2, showing several cavities. FIG. 18. A section through an animal half embryo, showing the unorganized nature of the embryo. FIG. 19. A section through an anterior animal quarter embryo, 2a4.2, showing epidermal vesicle formation. FIG. 20. A section of a 2/14.1 + 264.2 embryo through the induced cerebral vesicle and otolith. FIG. 21. A section through a 2.44.1 + 264.2 embryo, showing the induced pigment spot. FIG. 22. A section through a 2a4.2 + 1^15.2 embryo. The otolith is attached to the cup of cells which arose from the 2a4.2. Inserted in the concavity of the cup may be seen the plug of cells derived from the .45.2 cell. PIG. 23. The section passes through a cerebral vesicle containing a typical otolith in a 2a4.2 + 2.45.1 combination. FIG. 24. A tail section of an unoperated embryo. The central notochord is flanked by the rows of dark myofibrillae. PIG. 25. A tail section of a posterior three-quarter embryo, showing the noto- chord flanked by rows of dark myofibrillae. 15 19 20 I Ot, vt 4 ^ 21 ot V 22 23 PLATE III 226 S. MERYL ROSE The isolation experiments seem to indicate that factors or inductors necessary for the differentiation of other parts of the embryo are located in the anterior vegetal region. The evidence for this is the ab- sence of differentiation in embryos which lack this region and the higher degree of differentiation of embryos which contain the anterior vegetal material. Transplantations A more striking and positive demonstration of an inductor is ob- tained when the inductor region in combination with cells incapable of self-differentiation causes those cells to form a structure which neither the inducing nor the reacting cells would form in normal development. Combinations of 2^44.1 + 2M.2 from the eight-cell stage have led to the development of partial embryos possessing cerebral vesicles and sensory cells. Figures 20 and 21 are sections of two such embryos through the otolith region. The number of ,44.1 -f &4.2 combinations which produced embryos containing pigmented sensory cells was fifteen out of forty-six. In these embryos presumptive epidermis cells have replaced presumptive cerebral vesicle cells, and, in combination with inductor, have formed cerebral vesicles and the pigmented sensory cells. An attempt to determine the extent of the cerebral vesicle inductor in the anterior vegetal quadrant has been made. The ^45.1 and the A5.2 cells of the sixteen-cell stage and the A6A and ,46.3 and the A6.2 and ,46.4 cells of the thirty-two cell stage have been combined with animal cells of the eight-cell stage. Both the .45.1 cells and the A5.2 cells have induced cerebral vesicles and sensory cells. Figure 23 shows a well-formed otolith in a cerebral vesicle. This embryo arose from a 2a4.2 + 2,45.1 combination. Figure 22 is a section through a 2a4.2 + 1A5.2 embryo. It is of interest because it shows that the otolith formed from an animal cell which was in direct contact with ,45.2 derivatives. The animal cells are seen in the form of a cup with a plug of vegetal cells protruding from the concavity of the cup. A few combinations were made in which one member of the pair was stained with Nile Blue Sulphate. In five instances gastrulation was incomplete and in these the sensory cells formed on the surface at the boundary between the stained and unstained portions. The proximity of the inducing vegetal cells and the reacting animal cells suggests a direct transfer of inducing substance from the vegetal to the animal cells. The extent of the inductor in the thirty-two cell stage is less clear. One thirty-two cell embryo from which the 2,46.2 + 2,46.4 cells were EMBRYONIC INDUCTION IN ASCIDIA 227 removed produced a sensory pigment cell. This was the only operation of this type performed. In this embryo the only anterior vegetal cells present were derivatives of the 2A6.1 and 2.46.3 cells, presumptive for endoderm and mesenchyme. The presumptive notochord and spinal cord cells were removed. A few other operations testing for the presence of neural inductor in the .46.1 and ^46.3 derivatives were per- formed late in the operating season during September when sensory pigment was not forming in the operated embryos, or even in a number of the control embryos. The combination was .46.1 + ^46.3 + 2a4.2. Of four successful combinations, two showed evidence of neural in- vagination. One of these two contained a solid internal rod of neural type cells. Of four embryos resulting from ^46.2 + ^46.4 + 2a4.2 combinations, none showed any evidence of neural invagination. The negative cases are so few here and the criteria of neural differentiation so limited that our knowledge of the neural inducing ability of the ^46.2 and .46.4 cells remains uncertain. The ^46.1 and -46. 3 cells in the whole embryo give rise to the endodermal cells which directly underlie the cerebral vesicle. It is probably they, in normal develop- ment, which induce the cerebral vesicle. Twenty combinations of 254.1 -f- 2a4.2 gave no evidence of neural differentiation (Fig. 12). The embryos are very similar in appearance to posterior half embryos, 2B3 (Fig. 11). This result indicates that the neural inductor is limited to the anterior vegetal region and does not spread over the entire vegetal region. The extrinsic factors functioning in muscle differentiation will be described in a future paper. At present, it may be said that the pre- sumptive muscle cells, when they are part of a posterior half embryo, do not self-differentiate (Fig. 11). Neither do they differentiate when combined with anterior animal material, 254.1 + 2a4.2 (Fig. 12). Functional tail muscles do form, however, in posterior three-quarter embryos. The operations were performed in the thirty-two cell stage when the 2.45.1 + 2a5.3 cells were removed, leaving the 2a5.4 and 2y45.2 cells in combination with the posterior half of the embryo. Figure 11 is a photograph of a section of an unoperated embryo's tail, and Fig. 12 is a similar section of a tail of a posterior three-quarter embryo. Myofibrillae may be seen in both sections. Potency to Respond to Cerebral Inductor The relative potency to respond to the cerebral inductor has been found to differ in various parts of the embryo. Table II is a summary of the data. The normal number of sensory pigmented cells found in the cerebral vesicle of Styela is two. Rarely four appear. Blasto- 228 S. MERYL ROSE mere combinations which are predominantly of anterior materials regularly produce sensory cells. These cells, which form in normal development in the brain, may be considered to be evidence of the presence of neural differentiation, even though in many cases a neural tube has not formed. The number of sensory cells which developed TABLE II Reaction to Cerebral Inductor Combination Presumptive Value No. with Sensory Cells No. without Sensory Cells No. of Sensory Cells 2.43 Ep, CV, Not, SC, G, 22 2 1-9 Mes. 2o4.2+L45.ri 2a4.2 + 2/45.1J Ep, CV, Not, SC, G. 2 4 1-4 1-4 2a4.2 + 2/15.2l 2fl4.2 + L45.2J Ep, CV, Not, SC, G, Mes. 5 1 1 1-4 1 2a4.2+264.2 + 2/45.ll 2a4.2+264.2+L45.1J Ep, CV, Not, SC, G. 2 1 3 1-2 2 2a4.2 + 264.2 + 2/45.2\ 2a4.2+264.2+L45.2J Ep, CV, Not, SC, G, Mes. 3 1 1 2 1^ 2 2/13 + 154.1 Ep, CV, Not, SC, G, Mes, Mus. 3 2 2-3 164.2+244.1" 3 1-2 264.2 + 2,44.1 7 35 1-2 264.2 + 1/44.1 - Ep, Not, SC, G, Mes. 3 6 1-2 464.2+2,44.1 1 2 564.2+244.L 1 1 264.2 + 1,45.1) 264.2 + 2/15. 1J Ep, Not, SC, G. 1 3 - 264.2 + 1,45.21 1 264.2+2/15.2 Ep, Not, SC, G, Mes. 3 - 164.2 + 2,45.2] 1 1 254.1 + 264.2 + 1/14.1 Ep, Not, SC, G, Mes, Mus. 8 CV, cerebral vesicle; Ep, epidermis; G, gut; Mes, mesenchyme; Mus, muscle; Not, notochord; SC, spinal cord. in the anterior embryos was often greater than two and the amount of pigment was greater than in whole tadpoles. The numbers of pigment cells range from one to nine, the average being 3.8 for the anterior half embryos. The a4.2 + .45.1 or .45.2 combinations also regularly pro- duce neural tissue. Supernumerary sensory cells may also appear in EMBRYONIC INDUCTION IN ASCIDIA 229 these embryos. The sensory cell production in a4.2 + ^45.1 or ^45.2 embryos may be contrasted with that of embryos whose animal ma- terial comes from the posterior region, 64.2 -f- .45.1 or ^45.2. The a4.2 material has responded positively in twelve of thirteen cases, whereas the 64.2 material gave a negative response in eight of nine cases. This comparison is of embryos from the same batches of eggs. The response of posterior animal cells is somewhat better when the inductor cells are the .44.1 cells of the eight-cell stage. In this case both .45.1 and ^45.2 materials are represented. The positive responses with 64.2 + .44.1 were fifteen of fifty-six. This is in spite of the fact that most of the 64.2 + ^44.1 operations were performed before the introduction of the semi-sterile technique. Not only do anterior animal cells respond more often than posterior animal cells to the same inductor, but also the anterior cells produce more sensory structures. Never have the posterior animal cells pro- duced more than two sensory cells. The number is usually one. A further result obtained from the transplantation experiments is that the addition of posterior cells to combinations which alone would produce neural material decreases the frequency of its appearance. When the a4.2 cells alone were in combination with .45.1 or .45.2, twelve of thirteen embryos contained sensory cells. When 64.2 + a4.2 cells were host to .45.1 or ^45.2, only seven of thirteen produced sensory cells. Similarly, when the 254.1 material was added to a 264.2 + 1^44.1 combination, there were no sensory cells produced in eight cases. Alone the 264.2 + 1^44.1 combination had been shown to form sensory cells in three of nine cases. Although in some of the individual experiments the cases are too few, the combined data seem to allow the following conclusions: (1) Anterior animal cells have greater potency to form cerebral structures than do posterior animal cells. (2) Posterior cells tend to inhibit the formation of sensory structures in embryos containing competent materials. DISCUSSION The classical works of Conklin (1905&, 19056) on Styela demon- strated that early in development there is a segregation of ooplasmic materials. These visible cytoplasmic materials are correlated in normal development with particular embryonic organs or regions. However, some of these substances may be centrifugally displaced and come to lie in foreign organs (Conklin, 1931). In a sense, the segre- gation of visible ooplasmic materials is differentiation. Further, 230 S. MERYL ROSE isolated blastomeres differentiate in respect to cleavage patterns. But differentiation also includes the establishment of the various functional structures. The present work indicates that the anterior vegetal region is necessary for this latter type of differentiation. The earlier idea that the ascidian egg is a strict mosaic has been altered in recent years. Schmidt (1931) has found that lateral half embryos of dona intestinalis and Phallusia mammillata may sometimes form the normal three adhesive papillae. Cohen and Berrill (1936) obtained some rather normal appearing larvae from lateral half em- bryos of Ascidiella aspersa. They, however, interpreted the regulation as a mechanical regulation of an original mosaic pattern. Recently, von Ubisch (1938) has described a case in which two fused two-cell embryos of Ascidiella aspersa regulated to form a single individual. Dalcq (1932, 1938) has shown that lateral, or animal, or vegetal portions may be removed from the egg before fertilization without resulting depletion of organs in the larvae which develop from the egg. Reverberi (1931) obtained larvae very similar to normal larvae from fragments of fertilized Ciona eggs. The results of Dalcq and Reverberi plainly show that the egg is not a determined mosaic before completion of the first cleavage. Tung (1934) suggested the possibility that adhesive papillae and sensory cells might be dependent upon extrinsic factors, since they did not form in the isolated presumptive cells. The present work indi- cates that induction of organs is more general in the ascidian embryo. It appears that all cells outside of the anterior vegetal region differen- tiate dependently. This anterior vegetal region, presumptive for notochord, spinal cord, endoderm and some mesoderm, is similar in function to the corresponding region of the amphibian embryo, the organizer region. It is capable of self-differentiation and supplies necessary developmental factors to other regions. The great difference between amphibian dorsal embryos (Ruud, 1925) and ascidian anterior embryos is that the former regulate and form more than they would as parts of intact embryos, while the latter offer no evidence of regulation. The recent work of Reverberi (1937) demonstrates that both animal and vegetal materials must be present in egg fragments of Ciona intestinalis in order that the sensory cells may differentiate. Rever- beri's work and the present work suggest a possible interpretation. There are fundamental regional differences in the egg. Materials necessary for the differentiation of endoderm and notochord and for the production of inducing substances are in highest concentration in the anterior vegetal region. Materials which react with the cerebral inducing substances, or materials which produce the reacting sub- EMBRYONIC INDUCTION IN ASCIDIA 231 stances, are more concentrated in the animal region, especially the anterior animal region. The contiguity of original animal and vegetal regions established during gastrulation enables the interaction of anterior vegetal inducing substance or substances and the reacting animal material, which process leads to the differentiation of cerebral vesicle. CONCLUSIONS 1. Blastomeres from the animal region of the eight-cell stage are incapable of self-differentiation. 2. Posterior blastomeres of the four-cell stage are also unable to self-differentiate. 3. The anterior vegetal blastomeres of the eight-cell stage are capable of self-differentiation. 4. The anterior vegetal region is necessary for the differentiation of other regions. 5. The cerebral inductor is confined to the anterior vegetal region. 6. Presumptive epidermis may form brain under the influence of the inductor. 7. Anterior animal cells have greater potency to form cerebral structures than do posterior animal cells. 8. Posterior cells inhibit the formation of cerebral structures in embryos containing competent materials. LITERATURE CITED CHABRY, L., 1887. Contribution a 1'Embryologie Normale et Teratologique des Ascidies Simples. Jour, de I'Anat. et Physiol., 23: 167. CHILD, C. M., 1927. Developmental modification and elimination of the larval stage in the ascidian, Corella willmeriana. Jour. Morph., 44: 467. COHEN, A., AND N. J. BERRILL, 1936. The development of isolated blastomeres of the ascidian egg. Jour. Exper. Zool., 74: 91. CONKLIN, E. G., 1905o. The organization and cell lineage of the ascidian egg. Jour. Acad. Nat. Sci., Philadelphia, 13: 1. CONKLIN, E. G., 1905i. Mosaic development in ascidian eggs. Jour. Exper. Zool., 2: 145. CONKLIN, E. G., 1931. The development of centrifuged eggs of ascidians. Jour. Exper. Zool., 60: 1. DALCQ, A., 1932. Etudes des Localisation Germinales dans 1'Oeuf Vierge d'Ascidie par des Experiences de Merogonie. Arch, d'anat. Micros., 28: 223. DALCQ, A., 1938. Etude micrographique et quantitative de la Merogonie double chez Ascidiella scabra. Arch, de Biol., 49: 397. REVERBERI, G., 1931. Studi sperimentali sull 'uovo di Ascidie. PuU. Staz. Zool. Napoli, 11: 168. REVERBERI, G., 1937. Richerche sperimentali sulla struttura dell'uovo fecondato delle ascidie. Commentationes Pontif. Acad. Scient., 1: 135. 232 S. MERYL ROSE RUUD, G., 1925. Die Entwicklung isolierter Keimfragmente frtihester Stadien von Triton taeniatus. Roux' Arch., 105: 209. SCHMIDT, G. A., 1931. Die Entwicklung der Palpen bei Ascidienhalbeilarven. Arch. Zool. Hal. (Torino), 16: 490. SMITH, HOMER W., AND G. H. A. CLOWES, 1924. The influence of hydrogen ion concentration on the development of normally fertilized Arbacia and Asterias eggs. Biol. Bull., 47: 323. TUNG, Ti-CHOW, 1934. Recherches sur les potentialites des Blastomeres chez Ascidiella scabra. Arch, d'anat. Micros., 30: 381. VON UBISCH, L., 1938. Uber Keimschmelzungen an Ascidiella aspersa. Roux' Arch., 138: 18. ANDROGENETIC DEVELOPMENT OF THE EGG OF RANA PIPIENS 1 K. R. PORTER (From the Biological Laboratories, Harvard University and the Department of Biology, Princeton University) INTRODUCTION The aim of the investigator in seeking to initiate androgenetic development is to remove or inactivate the female pronucleus, at the same time leaving undisturbed the male pronucleus (if it is within the egg), the cytoplasm, and conditions essential for activation and first cleavage. To achieve this, especially by mechanical means, it is important that the egg be large, that the position of the egg chromatin be detectable, and that development proceed under laboratory condi- tions. It is, therefore, not surprising that the amphibian egg has been generally used. G. Hertwig, in 1911, treated the eggs of Rana fusca with radium emanations, then fertilized them, and obtained androgenetic develop- ment for what appears to be the first time. Since then a variety of methods have been used to remove or inactivate the egg nucleus (see below). These have been applied to various European species of frogs (G. Hertwig, 1911; P. Hertwig, 1923; Dalcq, 1932) and toads (G. Hertwig, 1913; P. Hertwig, 1923), to various species of Triton (P. Hertwig, 1916, 1923; G. Hertwig, 1927; Curry, 1931, 1936; Baltzer, 1933; Baltzer and de Roche, 1936; Hadorn, 1934) and to one American species, Triturus viridescens (Kaylor, 1937). None of these experiments has produced an adult haploid. In general, with androgenetic haploids as with haploids produced by parthenogenesis, gynogenesis and merogany, development ceases after a few days or in some cases a few weeks, is always abnormal, and where it continues to the larval stages produces an animal which is inactive and edematous. Despite their abnormalities, these haploids offer numerous possi- bilities for the study of nucleo-cytoplasmic relationships. Indeed, the abnormalities in themselves are not without interest, for an experi- 1 Part of data previously presented in thesis submitted to the faculty of Harvard University in partial fulfilment of the requirements for the degree of Doctor of Philosophy, June, 1938; part of data from experiments performed during tenure of National Research Fellowship at Princeton University. 233 234 K. R. PORTER mental demonstration of their cause should throw considerable light on the problems of differentiation. To be most serviceable as an experimental material, it seems essential that the haploids and the methods by which they are produced should possess certain positive characteristics. Their development should be fairly normal and con- tinue to an advanced stage of differentiation; the peculiarities of haploid development should be uniformly displayed by all animals; the haploid nuclear condition should remain unchanged; and the operative technique should be simple, effective, and capable of pro- ducing relatively large numbers. Haploids produced from eggs of various species of amphibia and by a variety of methods have satisfied these criteria to varying degrees, in no case perfectly. In view of this fact it is important to experiment further with new materials and methods. The report which follows presents the results of such experiments. An effective technique for the removal of the egg chromatin from the egg of Rana pipiens is described; the development which results from these operated eggs is described and compared with the normal diploid ; it is shown that the great majority of these animals develop as haploids; and certain cytological observations are presented which are of possible importance in explaining the abnormalities of haploid development. I should like to express my sincere gratitude to Professor Leigh Hoadley for his aid and advice during early investigations of this material. I am also indebted to Professor G. Fankhauser for valued suggestions in more recent studies. MATERIALS AND METHODS The eggs of the frog, Rana pipiens, secured from the state of Vermont were used in these experiments. Ovulation was induced by injecting water extracts of the anterior lobe of the frog pituitary following in general the method described by Rugh (1934). Such eggs when inseminated usually give a high percentage of fertilization and since the development which follows is perfectly normal there is little reason for considering the eggs so obtained as inadequate for experimental purposes. The operation, which results in the removal of the maternal chromatin, is simple and effective. Since it is in part original to these investigations and since its successful application depends on an under- standing of events taking place within the egg, a rather complete description follows. At the time of insemination the egg o